Anti-AVB6 antibodies and antibody-drug conjugates

ABSTRACT

Provided are novel anti-αvβ6 antibodies and antibody-drug conjugates and methods of using such anti-αvβ6 antibodies and antibody-drug conjugates to treat cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional App. No.62/943,959 filed Dec. 5, 2019 and U.S. Provisional App. No. 63/012,584filed Apr. 20, 2020, each of which is incorporated by reference in itsentirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file namedAVB6-00212_ST25 created on Nov. 16, 2020 and containing 52 KB, which ishereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to novel anti-αvβ6 antibodies andantibody-drug conjugates and methods of using such anti-αvβ6 antibodiesand antibody-drug conjugates to treat cancer.

BACKGROUND

αvβ6, which is also known as alpha-v beta-6, is a cell adhesion receptorthat binds extracellular matrix proteins such as fibronectin. αvβ6 iscomposed of an alpha v subunit and a beta 6 subunit, and is upregulatedin multiple cancers, including non-small cell lung cancer (NSCLC).

NSCLC is the most common type of lung cancer. In the past year, over200,000 people were diagnosed with lung cancer, which is the leadingcause of cancer death. Therefore, there is a need for improvedtreatments for NSCLC.

All references cited herein, including patent applications, patentpublications, and scientific literature, are herein incorporated byreference in their entirety, as if each individual reference werespecifically and individually indicated to be incorporated by reference.

SUMMARY

Provided herein are anti-αvβ6 antibodies and αvβ6-directed antibody-drugconjugates (ADCs). Also provided herein are methods of usinganti-αvβ6-directed antibodies and ADCs to treat αvβ6-expressingdisorders, including cancers. Preferred anti-αvβ6 antibodies compriseheavy chain CDR sequences of SEQ ID NOs: 31, 32, and 33 and light chainCDR sequences of SEQ ID NOs: 37, 42, and 39, as determined by Kabatnumbering.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a LAP blockade ELISA assay with variousanti-αvβ6 antibodies.

FIG. 2 shows the results of saturation binding studies on 293F cellsexpressing human and cyno αvβ6 with the murine antibody clone 2A2(referred to as m2A2).

FIG. 3 shows an alignment of the amino acid sequences of the parentalmurine mAb (referred to as m2A2) heavy chain variable region with selecthuman germline acceptor (referred to as hIGHV1-46/HJ4) and humanized 2A2heavy chain variants. Asterisks represent CDRs as determined by Kabat,and crosses represent CDRs as determined by IMGT.

FIG. 4 shows an alignment of the amino acid sequences of the parentalmurine mAb (referred to as m2A2) light chain variable region with selecthuman germline acceptor (referred to as hIGKV1D-33/KJ2) and humanized2A2 light chain variants. Asterisks represent CDRs as determined byKabat, and crosses represent CDRs as determined by IMGT.

FIG. 5 shows the results of competition binding studies on 293F cellsexpressing human αvβ6 with humanized antibodies having the LA and LBlight chains and the parental murine and chimeric antibodies (referredto as m2A2 and c2A2, respectively).

FIG. 6 shows the results of competition binding studies on 293F cellsexpressing human αvβ6 with humanized antibodies having the HA and HCheavy chains and the parental murine and chimeric antibodies (referredto as m2A2 and c2A2, respectively).

FIG. 7 shows the repeated results of competition binding studies on 293Fcells expressing human αvβ6 with a subset of humanized antibodies andthe parental murine antibody (referred to as m2A2).

FIG. 8 shows the results of saturation binding studies on 293F cellsexpressing human and cyno αvβ6 with the h2A2 HCLG humanized antibody andthe parental murine antibody (referred to as m2A2).

FIG. 9 shows the results of competition binding studies on 293F cellsexpressing human and cyno αvβ6 with HCLG humanized antibody and ADC.

FIG. 10 shows the results of αvβ6 specific binding studies by ELISA tohuman αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 with the h2A2 HCLG humanizedantibody.

FIG. 11 shows that h2A2 HCLG anti-αvβ6 vcMMAE ADC exhibits in vitrocytotoxicity against αvβ6 expressing cancer cell lines.

FIG. 12 shows the results of a xenograft study of the Detroit 562 headand neck cancer cell line in nude mice. The dose and schedule areindicated on the figure.

FIG. 13 shows the results of a xenograft study of the HPAFII pancreaticcancer cell line in nude mice. The dose and schedule are indicated onthe figure.

FIG. 14 shows the results of a xenograft study of the BxPC-3 pancreaticcancer cell line in nude mice. The dose and schedule are indicated onthe figure.

FIG. 15 shows the results of a xenograft study of the SW780 bladdercancer cell line in nude mice. The dose and schedule are indicated onthe figure.

FIG. 16 shows the results of a PDX study in six NSCLC cell lines in nudemice. ADCs were dosed at 3 mg/kg q7dx3.

FIG. 17 shows the results of a PDX study in six ovarian carcinoma celllines in nude mice. ADCs were dosed at 5 mg/kg q7dx3.

FIG. 18 shows the results of a comparison between h2A2 HCLG and h15H3 intwo cell line xenograft models. ADCs were dosed once at 3 mg/kg.

DETAILED DESCRIPTION I. Definitions

In order that the present disclosure can be more readily understood,certain terms are first defined. As used in this application, except asotherwise expressly provided herein, each of the following terms shallhave the meaning set forth below. Additional definitions are set forththroughout the application.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. The headings provided herein are notlimitations of the various aspects of the disclosure, which can be hadby reference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification in its entirety.

The terms “αvβ6,” “αvb6,” “alpha-v beta-6,” or “P6” are usedinterchangeably herein, and, unless specified otherwise, include anyvariants, isoforms and species homologs of human αvβ6 which aregenerally expressed by cells or expressed on cells transfected with theαvβ6 gene.

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four inter-connected by disulfide bonds. The structure ofimmunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as V_(H) or VH) and a heavychain constant region (C_(H) or CH). The heavy chain constant regiontypically is comprised of three domains, C_(H)1, C_(H)2, and C_(H)3. Theheavy chains are generally inter-connected via disulfide bonds in theso-called “hinge region.” Each light chain typically is comprised of alight chain variable region (abbreviated herein as V_(L) or VL) and alight chain constant region (C_(L) or CL). The light chain constantregion typically is comprised of one domain, C_(L). The CL can be of κ(kappa) or λ, (lambda) isotype. The terms “constant domain” and“constant region” are used interchangeably herein. An immunoglobulin canderive from any of the commonly known isotypes, including but notlimited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are alsowell known to those in the art and include but are not limited to humanIgG1, IgG2, IgG3 and IgG4. “Isotype” refers to the antibody class orsubclass (e.g., IgM or IgG1) that is encoded by the heavy chain constantregion genes.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable regions of the heavy chain and lightchain (V_(H) and V_(L), respectively) of a native antibody may befurther subdivided into regions of hypervariability (or hypervariableregions, which may be hypervariable in sequence and/or form ofstructurally defined loops), also termed complementarity-determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). The terms “complementarity determiningregions” and “CDRs,” synonymous with “hypervariable regions” or “HVRs”are known in the art to refer to non-contiguous sequences of amino acidswithin antibody variable regions, which confer antigen specificityand/or binding affinity. In general, there are three CDRs in each heavychain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in eachlight chain variable region (CDR-L1, CDR-L2, CDR-L3). “Frameworkregions” and “FR” are known in the art to refer to the non-CDR portionsof the variable regions of the heavy and light chains. In general, thereare four FRs in each full-length heavy chain variable region (FR-H1,FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chainvariable region (FR-L1, FR-L2, FR-L3, and FR-L4). Within each V_(H) andV_(L), three CDRs and four FRs are typically arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4 (See also Chothia and Lesk J. Mot. Biol., 195,901-917 (1987)).

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule, a fragment of an immunoglobulin molecule,or a derivative of either thereof, which has the ability to specificallybind to an antigen under typical physiological conditions with ahalf-life of significant periods of time, such as at least about 30 min,at least about 45 min, at least about one hour (h), at least about twohours, at least about four hours, at least about eight hours, at leastabout 12 hours (h), about 24 hours or more, about 48 hours or more,about three, four, five, six, seven or more days, etc., or any otherrelevant functionally-defined period (such as a time sufficient toinduce, promote, enhance, and/or modulate a physiological responseassociated with antibody binding to the antigen and/or time sufficientfor the antibody to recruit an effector activity). The variable regionsof the heavy and light chains of the immunoglobulin molecule contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies (Abs) may mediate the binding of the immunoglobulin tohost tissues or factors, including various cells of the immune system(such as effector cells) and components of the complement system such asC1q, the first component in the classical pathway of complementactivation. An antibody may also be a bispecific antibody, diabody,multispecific antibody or similar molecule.

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules that are recombinantly produced with a single primaryamino acid sequence, are produced from mouse B-cell fusions. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope. Accordingly, the term “humanmonoclonal antibody” refers to antibodies displaying a single bindingspecificity which have variable and constant regions derived from humangermline immunoglobulin sequences. The human monoclonal antibodies maybe generated by a hybridoma which includes a B cell obtained from atransgenic or transchromosomal non-human animal, such as a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene, fused to an immortalized cell.

An “isolated antibody” refers to an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that binds specifically to αvβ6 is substantially freeof antibodies that bind specifically to antigens other than αvβ6). Anisolated antibody that binds specifically to αvβ6 can, however, havecross-reactivity to other antigens, such as αvβ6 molecules fromdifferent species. Moreover, an isolated antibody can be substantiallyfree of other cellular material and/or chemicals. In one embodiment, anisolated antibody includes an antibody conjugate attached to anotheragent (e.g., small molecule drug). In some embodiments, an isolatedanti-αvβ6 antibody includes a conjugate of an anti-αvβ6 antibody with asmall molecule drug (e.g., MMAE or MMAF).

A “human antibody” (HuMAb) refers to an antibody having variable regionsin which both the FRs and CDRs are derived from human germlineimmunoglobulin sequences. Furthermore, if the antibody contains aconstant region, the constant region also is derived from human germlineimmunoglobulin sequences. The human antibodies of the disclosure caninclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody,” as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. The terms “human antibodies” and “fully human antibodies” andare used synonymously.

The term “humanized antibody” as used herein, refers to a geneticallyengineered non-human antibody, which contains human antibody constantdomains and non-human variable domains modified to contain a high levelof sequence homology to human variable domains. This can be achieved bygrafting of the six non-human antibody complementarity-determiningregions (CDRs), which together form the antigen binding site, onto ahomologous human acceptor framework region (FR) (see WO92/22653 andEP0629240). In order to fully reconstitute the binding affinity andspecificity of the parental antibody, the substitution of frameworkresidues from the parental antibody (i.e. the non-human antibody) intothe human framework regions (back-mutations) may be required. Structuralhomology modeling may help to identify the amino acid residues in theframework regions that are important for the binding properties of theantibody. Thus, a humanized antibody may comprise non-human CDRsequences, primarily human framework regions optionally comprising oneor more amino acid back-mutations to the non-human amino acid sequence,and fully human constant regions. Optionally, additional amino acidmodifications, which are not necessarily back-mutations, may be appliedto obtain a humanized antibody with preferred characteristics, such asaffinity and biochemical properties.

The term “chimeric antibody” as used herein, refers to an antibodywherein the variable region is derived from a non-human species (e.g.derived from rodents) and the constant region is derived from adifferent species, such as human. Chimeric antibodies may be generatedby antibody engineering. “Antibody engineering” is a term used genericfor different kinds of modifications of antibodies, and which is awell-known process for the skilled person. In particular, a chimericantibody may be generated by using standard DNA techniques as describedin Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, NewYork: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, the chimericantibody may be a genetically or an enzymatically engineered recombinantantibody. It is within the knowledge of the skilled person to generate achimeric antibody, and thus, generation of the chimeric antibodyaccording to the present invention may be performed by other methodsthan described herein. Chimeric monoclonal antibodies for therapeuticapplications are developed to reduce antibody immunogenicity. They maytypically contain non-human (e.g. murine) variable regions, which arespecific for the antigen of interest, and human constant antibody heavyand light chain domains. The terms “variable region” or “variabledomains” as used in the context of chimeric antibodies, refers to aregion which comprises the CDRs and framework regions of both the heavyand light chains of the immunoglobulin.

An “anti-antigen antibody” refers to an antibody that binds to theantigen. For example, an anti-αvβ6 antibody is an antibody that binds tothe antigen αvβ6.

An “antigen-binding portion” or antigen-binding fragment” of an antibodyrefers to one or more fragments of an antibody that retain the abilityto bind specifically to the antigen bound by the whole antibody.Examples of antibody fragments (e.g., antigen-binding fragment) includebut are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies;linear antibodies; single-chain antibody molecules (e.g. scFv); andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Percent (%) sequence identity” with respect to a reference polypeptidesequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, SnapGene Align, or ClustalWBioEdit software. Those skilled in the art can determine appropriateparameters for aligning sequences, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. For example, the % sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % sequence identity to, with, or against a givenamino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence in that program's alignment of A and B, and where Y isthe total number of amino acid residues in B. It will be appreciatedthat where the length of amino acid sequence A is not equal to thelength of amino acid sequence B, the % sequence identity of A to B willnot equal the % sequence identity of B to A.

As used herein, the terms “binding”, “binds” or “specifically binds” inthe context of the binding of an antibody to a pre-determined antigentypically is a binding with an affinity corresponding to a K_(D) ofabout 10⁻⁶ M or less, e.g. 10⁻⁷ M or less, such as about 10⁻⁸ M or less,such as about 10⁻⁹ M or less, about 10⁻¹⁰ M or less, or about 10⁻¹¹M oreven less when determined by for instance BioLayer Interferometry (BLI)technology in a Octet HTX instrument using the antibody as the ligandand the antigen as the analyte, and wherein the antibody binds to thepredetermined antigen with an affinity corresponding to a K_(D) that isat least ten-fold lower, such as at least 100-fold lower, for instanceat least 1,000-fold lower, such as at least 10,000-fold lower, forinstance at least 100,000-fold lower than its K_(D) of binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely related antigen. The amount with which the K_(D) ofbinding is lower is dependent on the K_(D) of the antibody, so that whenthe K_(D) of the antibody is very low, then the amount with which theK_(D) of binding to the antigen is lower than the K_(D) of binding to anon-specific antigen may be at least 10,000-fold (that is, the antibodyis highly specific).

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction.Affinity, as used herein, and K_(D) are inversely related, that is thathigher affinity is intended to refer to lower K_(D), and lower affinityis intended to refer to higher K_(D).

The term “ADC” refers to an antibody-drug conjugate, which in thecontext of the present invention refers to an anti-αvβ6 antibody, whichis coupled to a drug moiety (e.g., MMAE or MMAF) as described in thepresent application.

The abbreviations “vc” and “val-cit” refer to the dipeptidevaline-citrulline.

The abbreviation VKG refers to the tripeptide linkervaline-lysine-glycine.

The abbreviation “PAB” refers to the self-immolative spacer:

The abbreviation “MC” refers to the stretcher maleimidocaproyl:

The abbreviation “MP” refers to the stretcher maleimidopropionyl:

“PEG Unit” as used herein is an organic moiety comprised of repeatingethylene-oxy subunits (PEGs or PEG subunits) and may be polydisperse,monodisperse or discrete (i.e., having discrete number of ethylene-oxysubunits). Polydisperse PEGs are a heterogeneous mixture of sizes andmolecular weights whereas monodisperse PEGs are typically purified fromheterogeneous mixtures and are therefore provide a single chain lengthand molecular weight. Preferred PEG Units comprises discrete PEGs,compounds that are synthesized in step-wise fashion and not via apolymerization process. Discrete PEGs provide a single molecule withdefined and specified chain length.

The PEG Unit provided herein comprises one or multiple polyethyleneglycol chains, each comprised of one or more ethyleneoxy subunits,covalently attached to each other. The polyethylene glycol chains can belinked together, for example, in a linear, branched or star shapedconfiguration. Typically, at least one of the polyethylene glycol chainsprior to incorporation into a camptothecin conjugate is derivatized atone end with an alkyl moiety substituted with an electrophilic group forcovalent attachment to the carbamate nitrogen of a methylene carbamateunit (i.e., represents an instance of R). Typically, the terminalethyleneoxy subunit in each polyethylene glycol chains not involved incovalent attachment to the remainder of the Linker Unit is modified witha PEG Capping Unit, typically an optionally substituted alkyl such as—CH₃, CH₂CH₃ or CH₂CH₂CO₂H. A preferred PEG Unit has a singlepolyethylene glycol chain with 2 to 24 —CH₂CH₂O— subunits covalentlyattached in series and terminated at one end with a PEG Capping Unit.

A “cancer” refers to a broad group of various diseases characterized bythe uncontrolled growth of abnormal cells in the body. A “cancer” or“cancer tissue” can include a tumor. Unregulated cell division andgrowth results in the formation of malignant tumors that invadeneighboring tissues and can also metastasize to distant parts of thebody through the lymphatic system or bloodstream. Following metastasis,the distal tumors can be said to be “derived from” the pre-metastasistumor.

The term “antibody-dependent cellular cytotoxicity” ADCC, is a mechanismfor inducing cell death that depends upon the interaction ofantibody-coated target cells with immune cells possessing lytic activity(also referred to as effector cells). Such effector cells includenatural killer cells, monocytes/macrophages and neutrophils. Theeffector cells attach to an Fe effector domain(s) of Ig bound to targetcells via their antigen-combining sites. Death of the antibody-coatedtarget cell occurs as a result of effector cell activity.

The term “antibody-dependent cellular phagocytosis”, or ADCP, refers tothe process by which antibody-coated cells are internalized, either inwhole or in part, by phagocytic immune cells (e.g., macrophages,neutrophils and dendritic cells) that bind to an Fe effector domain(s)of Ig.

The term “complement-dependent cytotoxicity”, or CDC, refers to amechanism for inducing cell death in which an Fc effector domain(s) of atarget-bound antibody activates a series of enzymatic reactionsculminating in the formation of holes in the target cell membrane.Typically, antigen-antibody complexes such as those on antibody-coatedtarget cells bind and activate complement component Clq which in turnactivates the complement cascade leading to target cell death.Activation of complement may also result in deposition of complementcomponents on the target cell surface that facilitate ADCC by bindingcomplement receptors (e.g., CR3) on leukocytes.

A “cytostatic effect” refers to the inhibition of cell proliferation. A“cytostatic agent” refers to an agent that has a cytostatic effect on acell, thereby inhibiting the growth and/or expansion of a specificsubset of cells. Cytostatic agents can be conjugated to an antibody oradministered in combination with an antibody.

“Treatment” or “therapy” of a subject refers to any type of interventionor process performed on, or the administration of an active agent to,the subject with the objective of reversing, alleviating, ameliorating,inhibiting, slowing down, or preventing the onset, progression,development, severity, or recurrence of a symptom, complication,condition, or biochemical indicia associated with a disease. In someembodiments, the disease is cancer.

A “subject” includes any human or non-human animal. The term “non-humananimal” includes, but is not limited to, vertebrates such as non-humanprimates, sheep, dogs, and rodents such as mice, rats, and guinea pigs.In some embodiments, the subject is a human. The terms “subject” and“patient” and “individual” are used interchangeably herein.

An “effective amount” or “therapeutically effective amount” or“therapeutically effective dosage” of a drug or therapeutic agent is anyamount of the drug that, when used alone or in combination with anothertherapeutic agent, protects a subject against the onset of a disease orpromotes disease regression evidenced by a decrease in severity ofdisease symptoms, an increase in frequency and duration of diseasesymptom-free periods, or a prevention of impairment or disability due tothe disease affliction. The ability of a therapeutic agent to promotedisease regression can be evaluated using a variety of methods known tothe skilled practitioner, such as in human subjects during clinicaltrials, in animal model systems predictive of efficacy in humans, or byassaying the activity of the agent in in vitro assays.

By way of example for the treatment of tumors, a therapeuticallyeffective amount of an anti-cancer agent inhibits cell growth or tumorgrowth by at least about 10%, by at least about 20%, by at least about30%, by at least about 40%, by at least about 50%, by at least about60%, by at least about 70%, or by at least about 80%, by at least about90%, by at least about 95%, by at least about 96%, by at least about97%, by at least about 98%, or by at least about 99% in a treatedsubject(s) (e.g., one or more treated subjects) relative to an untreatedsubject(s) (e.g., one or more untreated subjects). In some embodiments,a therapeutically effective amount of an anti-cancer agent inhibits cellgrowth or tumor growth by 100% in a treated subject(s) (e.g., one ormore treated subjects) relative to an untreated subject(s) (e.g., one ormore untreated subjects).

In other embodiments of the disclosure, tumor regression can be observedand continue for a period of at least about 20 days, at least about 30days, at least about 40 days, at least about 50 days, or at least about60 days.

A therapeutically effective amount of a drug (e.g., anti-αvβ6antibody-drug conjugate) includes a “prophylactically effective amount,”which is any amount of the drug that, when administered alone or incombination with an anti-cancer agent to a subject at risk of developinga cancer (e.g., a subject having a pre-malignant condition) or ofsuffering a recurrence of cancer, inhibits the development or recurrenceof the cancer. In some embodiments, the prophylactically effectiveamount prevents the development or recurrence of the cancer entirely.“Inhibiting” the development or recurrence of a cancer means eitherlessening the likelihood of the cancer's development or recurrence, orpreventing the development or recurrence of the cancer entirely.

As used herein, “subtherapeutic dose” means a dose of a therapeuticcompound (e.g., an anti-αvβ6 antibody-drug conjugate) that is lower thanthe usual or typical dose of the therapeutic compound when administeredalone for the treatment of a hyperproliferative disease (e.g., cancer).

An “immune-related response pattern” refers to a clinical responsepattern often observed in cancer patients treated with immunotherapeuticagents that produce antitumor effects by inducing cancer-specific immuneresponses or by modifying native immune processes. This response patternis characterized by a beneficial therapeutic effect that follows aninitial increase in tumor burden or the appearance of new lesions, whichin the evaluation of traditional chemotherapeutic agents would beclassified as disease progression and would be synonymous with drugfailure. Accordingly, proper evaluation of immunotherapeutic agents canrequire long-term monitoring of the effects of these agents on thetarget disease.

By way of example, an “anti-cancer agent” promotes cancer regression ina subject. In some embodiments, a therapeutically effective amount ofthe drug promotes cancer regression to the point of eliminating thecancer. “Promoting cancer regression” means that administering aneffective amount of the drug, alone or in combination with ananti-cancer agent, results in a reduction in tumor growth or size,necrosis of the tumor, a decrease in severity of at least one diseasesymptom, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. In addition, the terms “effective” and “effectiveness” withregard to a treatment includes both pharmacological effectiveness andphysiological safety. Pharmacological effectiveness refers to theability of the drug to promote cancer regression in the patient.Physiological safety refers to the level of toxicity or other adversephysiological effects at the cellular, organ and/or organism level(adverse effects) resulting from administration of the drug.

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration that is at least the same as thetreatment duration, or at least 1.5, 2.0, 2.5, or 3 times longer thanthe treatment duration.

As used herein, “complete response” or “CR” refers to disappearance ofall target lesions; “partial response” or “PR” refers to at least a 30%decrease in the sum of the longest diameters (SLD) of target lesions,taking as reference the baseline SLD; and “stable disease” or “SD”refers to neither sufficient shrinkage of target lesions to qualify forPR, nor sufficient increase to qualify for PD, taking as reference thesmallest SLD since the treatment started.

As used herein, “progression free survival” or “PFS” refers to thelength of time during and after treatment during which the disease beingtreated (e.g., cancer) does not get worse. Progression-free survival mayinclude the amount of time patients have experienced a complete responseor a partial response, as well as the amount of time patients haveexperienced stable disease.

As used herein, “overall response rate” or “ORR” refers to the sum ofcomplete response (CR) rate and partial response (PR) rate.

As used herein, “overall survival” or “OS” refers to the percentage ofindividuals in a group who are likely to be alive after a particularduration of time.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,4,4′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

“Administering” or “administration” refer to the physical introductionof a therapeutic agent to a subject, using any of the various methodsand delivery systems known to those skilled in the art. Exemplary routesof administration for the anti-αvβ6 antibody-drug conjugate includeintravenous, intramuscular, subcutaneous, intraperitoneal, spinal orother parenteral routes of administration, for example by injection orinfusion (e.g., intravenous infusion). The phrase “parenteraladministration” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion, as well as in vivoelectroporation. A therapeutic agent can be administered via anon-parenteral route, or orally. Other non-parenteral routes include atopical, epidermal or mucosal route of administration, for example,intranasally, vaginally, rectally, sublingually or topically.Administration can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods.

The terms “baseline” or “baseline value” used interchangeably herein canrefer to a measurement or characterization of a symptom before theadministration of the therapy (e.g., an anti-αvβ6 antibody-drugconjugate as described herein) or at the beginning of administration ofthe therapy. The baseline value can be compared to a reference value inorder to determine the reduction or improvement of a symptom of aαvβ6-associated disease contemplated herein (e.g., cancer). The terms“reference” or “reference value” used interchangeably herein can referto a measurement or characterization of a symptom after administrationof the therapy (e.g., an anti-αvβ6 antibody-drug conjugate asdescribed). The reference value can be measured one or more times duringa dosage regimen or treatment cycle or at the completion of the dosageregimen or treatment cycle. A “reference value” can be an absolutevalue; a relative value; a value that has an upper and/or lower limit; arange of values; an average value; a median value: a mean value; or avalue as compared to a baseline value.

Similarly, a “baseline value” can be an absolute value; a relativevalue; a value that has an upper and/or lower limit; a range of values;an average value; a median value; a mean value; or a value as comparedto a reference value. The reference value and/or baseline value can beobtained from one individual, from two different individuals or from agroup of individuals (e.g., a group of two, three, four, five or moreindividuals).

The term “monotherapy” as used herein means that the anti-αvβ6antibody-drug conjugate is the only anti-cancer agent administered tothe subject during the treatment cycle. Other therapeutic agents,however, can be administered to the subject. For example,anti-inflammatory agents or other agents administered to a subject withcancer to treat symptoms associated with cancer, but not the underlyingcancer itself, including, for example inflammation, pain, weight loss,and general malaise, can be administered during the period ofmonotherapy.

An “adverse event” (AE) as used herein is any unfavorable and generallyunintended or undesirable sign (including an abnormal laboratoryfinding), symptom, or disease associated with the use of a medicaltreatment. A medical treatment can have one or more associated AEs andeach AE can have the same or different level of severity. Reference tomethods capable of “altering adverse events” means a treatment regimethat decreases the incidence and/or severity of one or more AEsassociated with the use of a different treatment regime.

A “serious adverse event” or “SAE” as used herein is an adverse eventthat meets one of the following criteria:

-   -   Is fatal or life-threatening (as used in the definition of a        serious adverse event, “life-threatening” refers to an event in        which the patient was at risk of death at the time of the event;        it does not refer to an event which hypothetically might have        caused death if it was more severe.    -   Results in persistent or significant disability/incapacity    -   Constitutes a congenital anomaly/birth defect    -   Is medically significant, i.e., defined as an event that        jeopardizes the patient or may require medical or surgical        intervention to prevent one of the outcomes listed above.        Medical and scientific judgment must be exercised in deciding        whether an AE is “medically significant”    -   Requires inpatient hospitalization or prolongation of existing        hospitalization, excluding the following: 1) routine treatment        or monitoring of the underlying disease, not associated with any        deterioration in condition; 2) elective or pre-planned treatment        for a pre-existing condition that is unrelated to the indication        under study and has not worsened since signing the informed        consent; and 3) social reasons and respite care in the absence        of any deterioration in the patient's general condition.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives. Asused herein, the indefinite articles “a” or “an” should be understood torefer to “one or more” of any recited or enumerated component.

The terms “about” or “comprising essentially of” refer to a value orcomposition that is within an acceptable error range for the particularvalue or composition as determined by one of ordinary skill in the art,which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” or “comprising essentially of” can mean within 1 ormore than 1 standard deviation per the practice in the art.Alternatively, “about” or “comprising essentially of” can mean a rangeof up to 20%. Furthermore, particularly with respect to biologicalsystems or processes, the terms can mean up to an order of magnitude orup to 5-fold of a value. When particular values or compositions areprovided in the application and claims, unless otherwise stated, themeaning of “about” or “comprising essentially of” should be assumed tobe within an acceptable error range for that particular value orcomposition.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” encompasses anddescribes “X.”

As described herein, any concentration range, percentage range, ratiorange, or integer range is to be understood to include the value of anyinteger within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated.

Various aspects of the disclosure are described in further detail in thefollowing subsections.

II. General

The invention provides antibodies that specifically bind αvβ6. Thepresent invention is based, in part, on the discovery that antibody-drugconjugates, including vcMMAE antibody-drug conjugates, targeted to αvβ6are particularly effective at killing αvβ6+ expressing cells. αvβ6 hasbeen shown to be expressed in a variety of cancers, including non-smallcell lung cancer (NSCLC) (squamous and adeno), head and neck cancer(including head and neck squamous carcinoma), esophageal cancer, breastcancer (including breast invasive carcinoma), ovarian cancer, bladdercancer (including urothelial carcinoma), skin cancer (squamous cellcarcinoma, or SCC), renal cancer (including renal clear cell, renalpapillary cell, and kidney chromophobe), cervical cancer, gastriccancer, prostate cancer (including prostate adenocarcinoma), endometrialcancer (including uterine carcinosarcoma and uterine corpusendometrial), rectum adenocarcinoma, thyroid carcinoma, colonadenocarcinoma, stomach adenocarcinoma, and pancreatic cancer (includingpancreatic adenocarcinoma).

III. Target Molecules

Unless otherwise indicated, αvβ6 refers to human αvβ6. An exemplary (36human sequence is assigned GenBank accession number AAA36122. Anexemplary av human sequence is assigned NCBI NP_002201.1.

IV. Antibodies of the Invention

The invention provides the murine 2A2 antibody, and chimeric, humanized,and human 2A2 antibodies.

The affinity of antibodies of the present invention (e.g., chimeric,humanized and human forms of the mouse 2A2 antibody) for human αvβ6 ispreferably equivalent to the affinity of mouse 2A2 antibody for humanαvβ6, greater than the affinity of mouse 2A2 antibody for human αvβ6 orwithin a factor of ten, within a factor of five, or within a factor oftwo weaker than that of the murine antibody 2A2 for human αvβ6. Onemethod of measuring affinity of an antibody for its target antigen is bydetermining an antibody's apparent dissociation constant. The presentinvention encompasses antibodies (e.g., chimeric, humanized and humanforms of the mouse 2A2 antibody) having an apparent dissociationconstant that is essentially the same as that of murine 2A2 (i.e.,within experimental error) as well as antibodies having an dissociationconstant lower or higher than that of murine antibody 2A2 for humanαvβ6. Chimeric, humanized and human 2A2 antibodies specifically bind tohuman αvβ6 in native form and/or recombinantly expressed from CHO cellsas does the mouse 2A2 antibody. Typically, chimeric, humanized and human2A2 anti-αvβ6 antibodies compete with murine 2A2 for binding to humanαvβ6.

Preferred antibodies of the invention inhibit cancer (e.g., growth ofcells, metastasis and/or lethality to the organisms) as shown oncancerous cells propagating in culture, in an animal model or clinicaltrial. Animal models can be formed by implanting αvβ6-expressing humantumor cell lines into appropriate immunodeficient rodent strains, e.g.,athymic nude mice or SCID mice. These tumor cell lines can beestablished in immunodeficient rodent hosts either as solid tumor bysubcutaneous injections or as disseminated tumors by intravenousinjections. Once established within a host, these tumor models can beapplied to evaluate the therapeutic efficacies of the anti-αvβ6antibodies or conjugated forms thereof as described in the Examples.

Generally, anti-αvβ6 antibodies and/or anti-αvβ6 antibody-drugconjugates of the disclosure bind αvβ6, e.g., human αvβ6, and exertcytostatic and cytotoxic effects on malignant cells, such as cancercells. Anti-αvβ6 antibodies of the disclosure are preferably monoclonal,and may be multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, and αvβ6 binding fragments of anyof the above. In some embodiments, the anti-αvβ6 antibodies of thedisclosure specifically bind αvβ6. The immunoglobulin molecules of thedisclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

In certain embodiments of the disclosure, the anti-αvβ6 antibodies areantigen-binding fragments (e.g., human antigen-binding fragments) asdescribed herein and include, but are not limited to, Fab, Fab′ andF(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a V_(L) orV_(H) domain. Antigen-binding fragments, including single-chainantibodies, may comprise the variable region(s) alone or in combinationwith the entirety or a portion of the following: hinge region, CH1, CH2,CH3 and CL domains. Also included in the present disclosure areantigen-binding fragments comprising any combination of variableregion(s) with a hinge region, CH1, CH2, CH3 and CL domains. In someembodiments, the anti-αvβ6 antibodies or antigen-binding fragmentsthereof are human, murine (e.g., mouse and rat), donkey, sheep, rabbit,goat, guinea pig, camelid, horse, or chicken.

The anti-αvβ6 antibodies of the present disclosure may be monospecific,bispecific, trispecific or of greater multi specificity. Multispecificantibodies may be specific for different epitopes of αvβ6 or may bespecific for both αvβ6 as well as for a heterologous protein.

Anti-αvβ6 antibodies of the present disclosure may be described orspecified in terms of the particular CDRs they comprise. The preciseamino acid sequence boundaries of a given CDR or FR can be readilydetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme);Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numberingscheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996),“Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme);Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains,” Dev CompImmunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger Aand Plückthun A, “Yet another numbering scheme for immunoglobulinvariable domains: an automatic modeling and analysis tool,” J Mol Biol,2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme); and Martin et al.,“Modeling antibody hypervariable loops: a combined algorithm,” PNAS,1989, 86(23):9268-9272, (“AbM” numbering scheme). The boundaries of agiven CDR may vary depending on the scheme used for identification. Insome embodiments, a “CDR” or “complementarity determining region,” orindividual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a givenantibody or region thereof (e.g., variable region thereof) should beunderstood to encompass a (or the specific) CDR as defined by any of theaforementioned schemes. For example, where it is stated that aparticular CDR (e.g., a CDR-H3) contains the amino acid sequence of acorresponding CDR in a given V_(H) or V_(L) region amino acid sequence,it is understood that such a CDR has a sequence of the corresponding CDR(e.g., CDR-H3) within the variable region, as defined by any of theaforementioned schemes. The scheme for identification of a particularCDR or CDRs may be specified, such as the CDR as defined by the Kabat,Chothia, AbM or IMGT method.

In an embodiment, the CDR sequences of the anti-αvβ6 antibodies and ofthe anti-αvβ6 antibody-drug conjugates described herein are according tothe Kabat numbering scheme. In another embodiment, the CDR sequences ofthe anti-αvβ6 antibodies and of the anti-αvβ6 antibody-drug conjugatesdescribed herein are according to the IMGT numbering scheme.

In one aspect, provided herein is an anti-αvβ6 antibody comprising aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) CDR-H1 comprising theamino acid sequence of SEQ ID NO:31, (ii) CDR-H2 comprising the aminoacid sequence of SEQ ID NO:32, and (iii) CDR-H3 comprising the aminoacid sequence of SEQ ID NO:33; and/or wherein the light chain variableregion comprises (i) CDR-L1 comprising the amino acid sequence of SEQ IDNO:37, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:42,and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:39,wherein the CDRs of the anti-αvβ6 antibody are defined by the Kabatnumbering scheme. In other embodiments, CDR-L1 comprises the amino acidsequence of SEQ ID NO: 40. In other embodiments, CDR-L2 comprises theamino acid sequence of SEQ ID NO: 38 or 41.

In one aspect, provided herein is an anti-αvβ6 antibody comprising aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) CDR-H1 comprising theamino acid sequence of SEQ ID NO:34, (ii) CDR-H2 comprising the aminoacid sequence of SEQ ID NO:35, and (iii) CDR-H3 comprising the aminoacid sequence of SEQ ID NO:36; and/or wherein the light chain variableregion comprises (i) CDR-L1 comprising the amino acid sequence of SEQ IDNO:43, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:44,and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO:45,wherein the CDRs of the anti-αvβ6 antibody are defined by the IMGTnumbering scheme.

An anti-αvβ6 antibody described herein may comprise any suitableframework variable domain sequence, provided that the antibody retainsthe ability to bind αvβ6 (e.g., human αvβ6). In some embodiments of theanti-αvβ6 antibodies described herein, the heavy and the light chainvariable domains comprise the amino acid sequences of SEQ ID NO: 6 andSEQ ID NO: 17, respectively. In some embodiments of the anti-αvβ6antibodies described herein, the heavy and light chains comprise theamino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 29, respectively.

In some embodiments, provided herein is an anti-αvβ6 antibody and/oranti-αvβ6 antibody-drug conjugate comprising a heavy chain variabledomain comprising an amino acid sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the amino acid sequence of SEQ ID NO:6. In certainembodiments, a heavy chain variable domain comprising an amino acidsequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acidsequence of SEQ ID NO:6 contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence and retains the ability to bind to an αvβ6 (e.g., human αvβ6).In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:6. In otherembodiments, a total of 3 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:6. In certain embodiments,substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 aminoacids) occur in regions inside the CDRs. In certain embodiments,substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 aminoacids) occur in regions outside the CDRs (i.e., in the FRs). In someembodiments, the anti-αvβ6 antibody comprises a heavy chain variabledomain sequence of SEQ ID NO:6 including post-translationalmodifications of that sequence.

In some embodiments, provided herein is an anti-αvβ6 antibody and/oranti-αvβ6 antibody-drug conjugate comprising a light chain variabledomain comprising an amino acid sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the amino acid sequence of SEQ ID NO:17. In certainembodiments, a light chain variable domain comprising an amino acidsequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acidsequence of SEQ ID NO:17 contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence and retains the ability to bind to an αvβ6 (e.g., human αvβ6).In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:17. In otherembodiments, a total of 1 to 2 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:17. In certain embodiments,substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 aminoacids) occur in regions outside the CDRs (i.e., in the FRs). In someembodiments, the anti-αvβ6 antibody comprises a light chain variabledomain sequence of SEQ ID NO:17 including post-translationalmodifications of that sequence.

There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM,having heavy chains designated α, δ, ε, γ and μ, respectively. The γ andα classes are further divided into subclasses e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. IgG1antibodies can exist in multiple polymorphic variants termed allotypes(reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any ofwhich are suitable for use in some of the embodiments herein. Commonallotypic variants in human populations are those designated by theletters a, f, n, z or combinations thereof. In any of the embodimentsherein, the antibody may comprise a heavy chain Fc region comprising ahuman IgG Fc region. In further embodiments, the human IgG Fc regioncomprises a human IgG1.

The antibodies of the invention also include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom binding to αvβ6 or from exerting a cytostatic or cytotoxic effecton HD cells. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, PEGylation, phosphylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No.6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. A preferred acceptor sequence for the heavy chain is thegermline V_(H) exon IGHV1-46 and for the J exon (J_(H)), exon IGHJ4. Forthe light chain, a preferred acceptor sequence is exon IGKV1D-33 and forthe J exon IGKJ2. Alternative preferred acceptor sequences for the heavychain include the J exons (J_(H)), IGHJ1, IGHJ2, IGHJ3, IGHJ5 or IGHJ6.Alternative preferred acceptor sequences for the light chain include Jexons IGKJ1, IGKJ3, IGKJ4 or IGKJ5. Thus, a humanized antibody is anantibody having some or all CDRs entirely or substantially from a donorantibody and variable region framework sequences and constant regions,if present, entirely or substantially from human antibody sequences.Similarly a humanized heavy chain has at least one, two and usually allthree CDRs entirely or substantially from a donor antibody heavy chain,and a heavy chain variable region framework sequence and heavy chainconstant region, if present, substantially from human heavy chainvariable region framework and constant region sequences. Similarly ahumanized light chain has at least one, two and usually all three CDRsentirely or substantially from a donor antibody light chain, and a lightchain variable region framework sequence and light chain constantregion, if present, substantially from human light chain variable regionframework and constant region sequences. Other than nanobodies and dAbs,a humanized antibody comprises a humanized heavy chain and a humanizedlight chain. A CDR in a humanized antibody is substantially from acorresponding CDR in a non-human antibody when at least 60%, 85%, 90%,95% or 100% of corresponding residues (as defined by Kabat) areidentical between the respective CDRs. The variable region frameworksequences of an antibody chain or the constant region of an antibodychain are substantially from a human variable region framework sequenceor human constant region respectively when at least 85%, 90%, 95% or100% of corresponding residues defined by Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a mouse antibody, they can also be made withless than all CDRs (e.g., at least 3, 4, or 5) CDRs from a mouseantibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos etal., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al.,Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology,164:1432-1441, 2000).

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region,

(3) otherwise interacts with a CDR region (e.g. is within about 6 A of aCDR region); or

(4) mediates interaction between the heavy and light chains.

Although the 2A2 antibody was identified as a mouse antibody, thepresent application also encompasses human 2A2 antibodies. By the term,“human 2A2 antibody” is meant an antibody that is derived from humanimmunoglobulin gene sequences and that has CDRs that are substantiallyidentical to those of murine 2A2 antibody and displays similarproperties, i.e., binding specificity to αvβ6. In some aspects, a human2A2 antibody comprises a heavy chain variable region that issubstantially identical to a heavy chain variable region describedherein and/or a light chain variable region that is substantiallyidentical to a light chain variable region described herein. In someembodiments, a 2A2 antibody of the present invention is not a humanantibody, e.g., a 2A2 antibody of the present invention is a murine,chimeric, or humanized antibody.

One aspect of the invention provides humanized forms of the mouseantibody 2A2. One such humanized variant of the mouse antibody 2A2 isdesignated HCLG. HCLG comprises a mature heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:6 and a mature lightchain variable region comprising the amino acid sequence of SEQ IDNO:17. Humanized antibodies of the invention include variants of theHCLG humanized antibody in which the humanized heavy chain maturevariable region shows at least 90%, 95% or 99% identity to SEQ ID NO: 6and the humanized light chain mature variable region shows at least 90%,95% or 99% sequence identity to SEQ ID NO:17. Preferably, in suchantibodies some or all of the backmutations in HCLG are retained. Inother words, at least 1, 2, 3, 4, 5, 6, 7, 8, or preferably all 9 ofheavy chain positions H2, H28, H48, H67, H69, H71, H73, H78, and H93 areoccupied by F, S, I, A, L, V, K, A, and T, respectively. Likewise,position L69 is preferably occupied by R, and L71 is preferably occupiedby Y. The CDR regions can be defined by any conventional definition(e.g., Chothia) but are preferably as defined by Kabat or IMGT. In oneembodiment, the humanized antibody comprises a heavy chain comprisingthe 3 CDRs of SEQ ID NO: 6 and variable region frameworks with at least95% identity to the variable region frameworks of SEQ ID NO: 6. Inanother embodiment, the humanized antibody comprises a light chaincomprising the 3 CDRs of SEQ ID NO: 17 and variable region frameworkswith at least 95% identity to variable region frameworks of SEQ ID NO:17. In a further embodiment, the humanized antibody comprises a heavychain comprising the 3 CDRs of SEQ ID NO: 6 and variable regionframeworks with at least 98% identity to the variable region frameworksof SEQ ID NO: 6, and a light chain comprising the 3 CDRs of SEQ ID NO:17, and variable region frameworks with at least 98% identity to thevariable region frameworks of SEQ ID NO: 17. In one embodiment, thehumanized antibody comprises a heavy chain comprising the 3 CDRs of SEQID NO: 6 and variable region frameworks with at least 99% identity tothe variable region frameworks of SEQ ID NO: 6. In another embodiment,the humanized antibody comprises a light chain comprising the 3 CDRs ofSEQ ID NO: 17 and variable region frameworks with at least 99% identityto variable region frameworks of SEQ ID NO: 17.

One possible variation is to substitute certain residues in the CDRs ofthe mouse antibody with corresponding residues from human CDRssequences, typically from the CDRs of the human acceptor sequences usedin designing the exemplified humanized antibodies. In some antibodiesonly part of the CDRs, namely the subset of CDR residues required forbinding, termed the SDRs, are needed to retain binding in a humanizedantibody. CDR residues not contacting antigen and not in the SDRs can beidentified based on previous studies (for example residues H60-H65 inCDR H2 are often not required), from regions of Kabat CDRs lying outsideChothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), bymolecular modeling and/or empirically, or as described in Gonzales etal., Mol. Immunol. 41: 863 (2004). In such humanized antibodies atpositions in which one or more donor CDR residues is absent or in whichan entire donor CDR is omitted, the amino acid occupying the positioncan be an amino acid occupying the corresponding position (by Kabatnumbering) in the acceptor antibody sequence. The number of suchsubstitutions of acceptor for donor amino acids in the CDRs to includereflects a balance of competing considerations. Such substitutions arepotentially advantageous in decreasing the number of mouse amino acidsin a humanized antibody and consequently decreasing potentialimmunogenicity. However, substitutions can also cause changes ofaffinity, and significant reductions in affinity are preferably avoided.Positions for substitution within CDRs and amino acids to substitute canalso be selected empirically.

Although not preferred other amino acid substitutions can be made, forexample, in framework residues not in contact with the CDRs, or evensome potential CDR-contact residues amino acids within the CDRs. Oftenthe replacements made in the variant humanized sequences areconservative with respect to the replaced HCLG amino acids. Preferably,replacements relative to HCLG (whether or not conservative) have nosubstantial effect on the binding affinity or potency of the humanizedmAb, that is, its ability to bind human αvβ6 and inhibit growth ofcancer cells.

Selection of Constant Region

The heavy and light chain variable regions of humanized antibodies canbe linked to at least a portion of a human constant region. The choiceof constant region depends, in part, whether antibody-dependentcell-mediated cytotoxicity, antibody dependent cellular phagocytosisand/or complement dependent cytotoxicity are desired. For example, humanisotopes IgG1 and IgG3 have strong complement-dependent cytotoxicity,human isotype IgG2 weak complement-dependent cytotoxicity and human.IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 alsoinduce stronger cell mediated effector functions than human IgG2 andIgG4. Light chain constant regions can be lambda or kappa. Antibodiescan be expressed as tetramers containing two light and two heavy chains,as separate heavy chains, light chains, as Fab, Fab′, F(ab′)2, and Fv,or as single chain antibodies in which heavy and light chain variabledomains are linked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype binds to a non-polymorphic region of a one or more otherisotypes.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004).

Exemplary substitution include the amino acid substitution of the nativeamino acid to a cysteine residue is introduced at amino acid position234, 235, 237, 239, 267, 298, 299, 326, 330, or 332, preferably an S239Cmutation in a human IgG1 isotype (US 20100158909). The presence of anadditional cysteine residue allows interchain disulfide bond formation.Such interchain disulfide bond formation can cause steric hindrance,thereby reducing the affinity of the Fc region-FcyR binding interaction.The cysteine residue(s) introduced in or in proximity to the Fc regionof an IgG constant region can also serve as sites for conjugation totherapeutic agents (i.e., coupling cytotoxic drugs using thiol specificreagents such as maleimide derivatives of drugs. The presence of atherapeutic agent causes steric hindrance, thereby further reducing theaffinity of the Fc region-FcyR binding interaction. Other substitutionsat any of positions 234, 235, 236 and/or 237 reduce affinity for Feyreceptors, particularly FcyRI receptor (see, e.g., U.S. Pat. Nos.6,624,821, 5,624,821.)

The in vivo half-life of an antibody can also impact on its effectorfunctions. The half-life of an antibody can be increased or decreased tomodify its therapeutic activities. FcRn is a receptor that isstructurally similar to MHC Class I antigen that non-covalentlyassociates with β2-microglobulin. FcRn regulates the catabolism of IgGsand their transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev.Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113).The IgG-FcRn interaction takes place at pH 6.0 (pH of intracellularvesicles) but not at pH 7.4 (pH of blood); this interaction enables IgGsto be recycled back to the circulation (Ghetie and Ward, 2000, Ann. Rev.Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113).The region on human IgG1 involved in FcRn binding has been mapped(Shields et al, 2001, J. Biol. Chem. 276:6591-604). Alaninesubstitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312,Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields etal, 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules harboring thesesubstitutions have longer serum half-lives. Consequently, these modifiedIgG1 molecules may be able to carry out their effector functions, andhence exert their therapeutic efficacies, over a longer period of timecompared to unmodified IgG1. Other exemplary substitutions forincreasing binding to FcRn include a Gln at position 250 and/or a Leu atposition 428. EU numbering is used for all position in the constantregion.

Oligosaccharides covalently attached to the conserved Asn297 areinvolved in the ability of the Fc region of an IgG to bind FcyR (Lund etal, 1996, J. Immunol. 157:4963-69; Wright and Morrison, 1999, TrendsBiotechnol. 15:26-31). Engineering of this glycoform on IgG cansignificantly improve IgG-mediated ADCC. Addition of bisectingN-acetylglucosamine modifications (Umana et al, 1999, Nat. Biotechnol.17:176-180; Davies et al, 2001, Biotech. Bioeng. 74:288-94) to thisglycoform or removal of fucose (Shields et al, 2002, J. Biol. Chem.277:26733-40; Shinkawa et al, 2003, J. Biol. Chem. 278:6591-604; Niwa etal., 2004, Cancer Res. 64:2127-33) from this glycoform are two examplesof IgG Fc engineering that improves the binding between IgG Fc and FcyR,thereby enhancing Ig-mediated ADCC activity.

A systemic substitution of solvent-exposed amino acids of human IgG1 Fcregion has generated IgG variants with altered FcyR binding affinities(Shields et al, 2001, J. Biol. Chem. 276:6591-604). When compared toparental IgG1, a subset of these variants involving substitutions atThr256/Ser298, Ser298/Glu333, Ser298/Lys334, or Ser298/Glu333 Lys334 toAla demonstrate increased in both binding affinity toward FcγR and ADCCactivity (Shields et al, 2001, J. Biol. Chem. 276:6591-604; Okazaki etal, 2004, J. Mol. Biol. 336:1239-49).

Complement fixation activity of antibodies (both Clq binding and CDCactivity) can be improved by substitutions at Lys326 and Glu333(Idusogie et al., 2001, J. Immunol. 166:2571-2575). The samesubstitutions on a human IgG2 backbone can convert an antibody isotypethat binds poorly to Clq and is severely deficient in complementactivation activity to one that can both bind Clq and mediate CDC(Idusogie et al, 2001, J. Immunol. 166:2571-75). Several other methodshave also been applied to improve complement fixation activity ofantibodies. For example, the grafting of an 18-amino acidcarboxyl-terminal tail piece of IgM to the carboxyl-termini of IgGgreatly enhances their CDC activity. This is observed even with IgG4,which normally has no detectable CDC activity (Smith et al, 1995, J.Immunol. 154:2226-36). Also, substituting Ser444 located close to thecarboxy-terminal of IgG 1 heavy chain with Cys induced tail-to-taildimerization of IgG 1 with a 200-fold increase of CDC activity overmonomeric IgG1 (Shopes et al, 1992, J. Immunol. 148:2918-22). Inaddition, a bispecific diabody construct with specificity for Clq alsoconfers CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

Complement activity can be reduced by mutating at least one of the aminoacid residues 318, 320, and 322 of the heavy chain to a residue having adifferent side chain, such as Ala. Other alkyl-substituted non-ionicresidues, such as Gly, He, Leu, or Val, or such aromatic non-polarresidues as Phe, Tyr, Trp and Pro in place of any one of the threeresidues also reduce or abolish Clq binding. Ser, Thr, Cys, and Met canbe used at residues 320 and 322, but not 318, to reduce or abolish Clqbinding activity.

Replacement of the 318 (Glu) residue by a polar residue may modify butnot abolish Clq binding activity. Replacing residue 297 (Asn) with Alaresults in removal of lytic activity but only slightly reduces (aboutthree fold weaker) affinity for Clq. This alteration destroys theglycosylation site and the presence of carbohydrate that is required forcomplement activation. Any other substitution at this site also destroysthe glycosylation site. The following mutations and any combinationthereof also reduce Clq binding: D270A, K322A, P329A, and P31 IS (see WO06/036291). The L234A/L235A mutation (or LALA mutation) also reduces C1qbinding, as well as FcyR binding.

Reference to a human constant region includes a constant region with anynatural allotype or any permutation of residues occupying polymorphicpositions in natural allotypes. Also, up to 1, 2, 5, or 10 mutations maybe present relative to a natural human constant region, such as thoseindicated above to reduce Fcgamma receptor binding or increase bindingto FcRN.

V. Expression of Recombinant Antibodies

Humanized antibodies are typically produced by recombinant expression.Recombinant polynucleotide constructs typically include an expressioncontrol sequence operably linked to the coding sequences of antibodychains, including naturally-associated or heterologous promoter regions.Preferably, the expression control sequences are eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and the collection andpurification of the crossreacting antibodies.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines (e.g., DG44), variousCOS cell lines, HeLa cells, HEK293 cells, L cells, andnon-antibody-producing myelomas including Sp2/0 and NS0. Preferably, thecells are nonhuman. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. Preferred expression control sequences are promoters derivedfrom endogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

VI. Nucleic Acids

The invention further provides nucleic acids encoding any of thehumanized heavy and light chains described above. Typically, the nucleicacids also encode a signal peptide fused to the mature heavy and lightchains. Coding sequences on nucleic acids can be in operable linkagewith regulatory sequences to ensure expression of the coding sequences,such as a promoter, enhancer, ribosome binding site, transcriptiontermination signal and the like. The nucleic acids encoding heavy andlight chains can occur in isolated form or can be cloned into one ormore vectors. The nucleic acids can be synthesized by for example, solidstate synthesis or PCR of overlapping oligonucleotides. Nucleic acidsencoding heavy and light chains can be joined as one contiguous nucleicacid, e.g., within an expression vector, or can be separate, e.g., eachcloned into its own expression vector.

In some aspects, also provided herein are nucleic acids encoding ananti-αvβ6 antibody or antigen-binding fragment thereof as describedherein. Further provided herein are vectors comprising the nucleic acidsencoding an anti-αvβ6 antibody or antigen-binding fragment thereof asdescribed herein. Further provided herein are host cells expressing thenucleic acids encoding an anti-αvβ6 antibody or antigen-binding fragmentthereof as described herein. Further provided herein are host cellscomprising the vectors comprising the nucleic acids encoding ananti-αvβ6 antibody or antigen-binding fragment thereof as describedherein.

The anti-αvβ6 antibodies described herein may be prepared by well-knownrecombinant techniques using well known expression vector systems andhost cells. In one embodiment, the antibodies are prepared in a CHO cellusing the GS expression vector system as disclosed in De la Cruz Edmundset al., 2006, Molecular Biotechnology 34; 179-190, EP216846, U.S. Pat.No. 5,981,216, WO 87/04462, EP323997, U.S. Pat. Nos. 5,591,639,5,658,759, EP338841, U.S. Pat. Nos. 5,879,936, and 5,891,693.

Monoclonal anti-αvβ6 antibodies described herein may e.g. be produced bythe hybridoma method first described by Kohler et al., Nature, 256, 495(1975), or may be produced by recombinant DNA methods. Monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in, for example, Clackson et al., Nature, 352,624-628 (1991) and Marks et al., J Mol, Biol., 222(3):581-597 (1991).Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B cells obtained from mice immunized withan antigen of interest, for instance in form of cells expressing theantigen on the surface, or a nucleic acid encoding an antigen ofinterest. Monoclonal antibodies may also be obtained from hybridomasderived from antibody-expressing cells of immunized humans or non-humanmammals such as rats, dogs, primates, etc.

VII. Antibody-Drug Conjugates

Anti-αvβ6 antibodies can be conjugated to cytotoxic or cytostaticmoieties (including pharmaceutically compatible salts thereof) to forman antibody drug conjugate (ADC). Particularly suitable moieties forconjugation to antibodies are cytotoxic agents (e.g., chemotherapeuticagents), prodrug converting enzymes, radioactive isotopes or compounds,or toxins (these moieties being collectively referred to as atherapeutic agent). For example, an anti-αvβ6 antibody can be conjugatedto a cytotoxic agent such as a chemotherapeutic agent, or a toxin (e.g.,a cytostatic or cytocidal agent such as, e.g., abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin).

An anti-αvβ6 antibody can be conjugated to a pro-drug converting enzyme.The pro-drug converting enzyme can be recombinantly fused to theantibody or chemically conjugated thereto using known methods. Exemplarypro-drug converting enzymes are carboxypeptidase G2, beta-glucuronidase,penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase,nitroreductase and carboxypeptidase A.

Techniques for conjugating therapeutic agents to proteins, and inparticular to antibodies, are well-known. (See, e.g., Arnon et al,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,”in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., AlanR. Liss, Inc., 1985); Hellstrom et al, “Antibodies For Drug Delivery,”in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc.,2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications (Pinchera et al. eds., 1985); “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled AntibodyIn Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection AndTherapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al,1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO89/12624.)

The therapeutic agent can be conjugated in a manner that reduces itsactivity unless it is cleaved off the antibody (e.g., by hydrolysis, byantibody degradation or by a cleaving agent). Such therapeutic agent isattached to the antibody with a cleavable linker that is sensitive tocleavage in the intracellular environment of the αvβ6-expressing cancercell but is not substantially sensitive to the extracellularenvironment, such that the conjugate is cleaved from the antibody whenit is internalized by the αvβ6-expressing cancer cell (e.g., in theendosomal or, for example by virtue of pH sensitivity or proteasesensitivity, in the lysosomal environment or in the caveolearenvironment).

Typically the ADC comprises a linker region between the therapeuticagent and the anti-αvβ6 antibody. As noted supra, typically, the linkeris cleavable under intracellular conditions, such that cleavage of thelinker releases the therapeutic agent from the antibody in theintracellular environment (e.g., within a lysosome or endosome orcaveolea). The linker can be, e.g., a peptidyl linker that is cleaved byan intracellular peptidase or protease enzyme, including a lysosomal orendosomal protease. Typically, the peptidyl linker is at least two aminoacids long or at least three amino acids long. Cleaving agents caninclude cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker,1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkersthat are cleavable by enzymes that are present in αvβ6-expressing cells.For example, a peptidyl linker that is cleavable by the thiol-dependentprotease cathepsin-B, which is highly expressed in cancerous tissue, canbe used (e.g., a linker comprising a Phe-Leu or a Gly-Phe-Leu-Glypeptide). Other such linkers are described, e.g., in U.S. Pat. No.6,214,345. In specific embodiments, the peptidyl linker cleavable by anintracellular protease comprises a Val-Cit linker or a Phe-Lys dipeptide(see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the Val-Cit linker). One advantage of usingintracellular proteolytic release of the therapeutic agent is that theagent is typically attenuated when conjugated and the serum stabilitiesof the conjugates are typically high.

The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysisat certain pH values. Typically, the pH-sensitive linker is hydrolyzableunder acidic conditions. For example, an acid-labile linker that ishydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone,thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or thelike) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805;5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123;Neville et al, 1989, Biol. Chem. 264: 14653-14661.) Such linkers arerelatively stable under neutral pH conditions, such as those in theblood, but are unstable at below pH 5.5 or 5.0, the approximate pH ofthe lysosome. In certain embodiments, the hydrolyzable linker is athioether linker (such as, e.g., a thioether attached to the therapeuticagent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).

Other linkers are cleavable under reducing conditions (e.g., a disulfidelinker). Disulfide linkers include those that can be formed using SATA(N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al, 1987, Cancer Res. 47:5924-5931;Wawrzynczak et al, In Immunoconjugates: Antibody Conjugates inRadioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press,1987. See also U.S. Pat. No. 4,880,935.)

The linker can also be a malonate linker (Johnson et al, 1995,Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al, 1995,Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog (Lau et al,1995, Bioorg-Med-Chem. 3(10):1305-12). The linker can also be a malonatelinker (Johnson et al, 1995, Anticancer Res. 15:1387-93), amaleimidobenzoyl linker (Lau et al, 1995, Bioorg-Med-Chem.3(10):1299-1304), or a 3′-N-amide analog (Lau et al, 1995,Bioorg-Med-Chem. 3(10):1305-12).

The linker also can be a non-cleavable linker, such as anmaleimido-alkylene- or maleimide-aryl linker that is directly attachedto the therapeutic agent (e.g., a drug). An active drug-linker isreleased by degradation of the antibody.

The linker can promote cellular internalization. The linker can promotecellular internalization when conjugated to the therapeutic agent (i.e.,in the milieu of the linker-therapeutic agent moiety of the ADC or ADCderivative as described herein). Alternatively, the linker can promotecellular internalization when conjugated to both the therapeutic agentand the anti-αvβ6 antibody (i.e., in the milieu of the ADC as describedherein).

The anti-αvβ6 antibody can be conjugated to the linker via a heteroatomof the antibody. These heteroatoms can be present on the antibody in itsnatural state or can be introduced into the antibody. In some aspects,the anti-αvβ6 antibody will be conjugated to the linker via a nitrogenatom of a lysine residue. In other aspects, the anti-αvβ6 antibody willbe conjugated to the linker via a sulfur atom of a cysteine residue. Thecysteine residue can be naturally-occurring or one that is engineeredinto the antibody. Methods of conjugating linkers and drug-linkers toantibodies via lysine and cysteine residues are known in the art.

Exemplary antibody-drug conjugates include auristatin basedantibody-drug conjugates (i.e., the drug component is an auristatindrug). Auristatins bind tubulin, have been shown to interfere withmicrotubule dynamics and nuclear and cellular division, and haveanticancer activity. Typically the auristatin based antibody-drugconjugate comprises a linker between the auristatin drug and theanti-αvβ6 antibody. The linker can be, for example, a cleavable linker(e.g., a peptidyl linker, a carbohydrate linker) or a non-cleavablelinker (e.g., linker released by degradation of the antibody).Auristatins include (but are not limited to) auristatin T, MMAF, andMMAE. The synthesis and structure of exemplary auristatins are describedin U.S. Pat. Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, andU.S. Pat. No. 7,968,687 each of which is incorporated herein byreference in its entirety and for all purposes.

Exemplary auristatin based antibody drug conjugates include vcMMAE (or1006), vcMMAF and mcMMAF antibody drug conjugates as shown below whereinp represents the drug load, Ab is an anti-αvβ6 antibody as describedherein and val-cit or “vc” represents the valine-citrulline dipeptide:

or a pharmaceutically acceptable salt thereof. The drug loading isrepresented by p, the number of drug-linker molecules per antibody.Referring to the αvβ6 targeted antibody-drug conjugates, the subscript prepresents the drug load and, depending on the context, can representthe number of molecules of drug-linker molecules attached to anindividual antibody molecule and as such, is an integer value, or canrepresent an average drug load and, as such, can be an integer ornon-integer value but is typically a non-integer value. An average drugload represents the average number of drug-linker molecules per antibodyin a population. Often, but not always, when we refer to an antibody,e.g., a monoclonal antibody, we are referring to a population ofantibody molecules. In a composition comprising a population ofantibody-drug conjugate molecules, the average drug load is an importantquality attribute as it determines the amount of drug that can bedelivered to a target cell. The percentage of unconjugated antibodymolecules in the composition is included in the average drug load value.

In preferred aspects of the present invention, the average drug loadwhen referring to a composition comprising a population of antibody-drugconjugate compounds is from 1 to about 16, preferably about 2 to about14, more preferably about 2 to about 10. In an embodiment, the DAR isfrom about 2 to about 5. In a further embodiment, the DAR is 4. Inanother embodiment, the DAR is from about 6 to about 10. In a furtherembodiment, the DAR is 8. The average number of drugs per antibody in apreparation may be characterized by conventional means such as massspectroscopy, HIC, ELISA assay, and HPLC. In some aspects, the anti-αvβ6antibody is attached to the drug-linker through a cysteine residue ofthe antibody. In some aspects, the cysteine residue is one that isengineered into the antibody. In other aspects, the cysteine residue isan interchain disulfide cysteine residue.

In some embodiments, incorporation of a polyethylene glycol polymer as aside chain into a cleavable β-glucuronide MMAE drug-linker providesantibody drug-conjugates with decreased plasma clearance and increasedantitumor activity in xenograft models as compared to a non-PEGylatedcontrol. Accordingly, particularly advantageous drug-linkers forattachment to the antibodies of the present invention are as follows informula V:

or a pharmaceutically acceptable salt thereof.

A preferred stereochemistry for such drug-linker is shown below informula Va:

or a pharmaceutically acceptable salt thereof wherein for formulas V andVa, Z represents an organic moiety having a reactive site capable ofreacting with a functional group on the antibody to form a covalentattachment thereto, n ranges from 8 to 36 and most preferably rangesfrom 8 to 14 (most preferably 12), R²¹ is a capping unit for thepolyethylene glycol moiety; preferably —CH₃ or —CH₂CH₂CO₂H.

A preferred Z moiety is a maleimido-containing moiety. Particularlypreferred Z moieties are shown in the drug-linkers below:

or a pharmaceutically acceptable salt thereof.

A preferred stereochemistry for such drug-linkers is shown below:

or a pharmaceutically acceptable salt thereof wherein for formulas VI,VIa, VII and VIIa, n ranges from 8 to 36 and most preferably ranges from8 to 14 (most preferably 12), R^(PR) is hydrogen or a protecting group,e.g., acid labile protecting group, e.g., BOC. R²¹ is a capping unit forthe polyethylene glycol moiety, preferably —CH₃ or —CH₂CH₂CO₂H.

As noted above, R^(PR) can be hydrogen or a protecting group. Protectivegroups as used herein refer to groups which selectively block, eithertemporarily or permanently, a reactive site in a multifunctionalcompound. A protecting group is a suitable protecting group when it iscapable of preventing or avoiding unwanted side-reactions or prematureloss of the protecting group under reaction conditions required toeffect desired chemical transformation elsewhere in the molecule andduring purification of the newly formed molecule when desired, and canbe removed under conditions that do not adversely affect the structureor stereochemical integrity of that newly formed molecule. Suitableamine protecting groups include acid-labile nitrogen protecting groups,including those provided by Isidro-Llobel et al. “Amino acid-protectinggroups” Chem. Rev. (2009) 109: 2455-2504. Typically, an acid-labilenitrogen-protecting group transforms a primary or secondary amino groupto its corresponding carbamate and includes t-butyl, allyl, and benzylcarbamates.

As noted above, R²¹ is a capping unit for the polyethylene glycolmoiety. As will be appreciated by the skilled artisan, polyethyleneglycol units can be terminally capped with a wide diversity of organicmoieties, typically those that are relatively non-reactive. Alkyl andsubstituted alkyl groups are preferred.

For the MMAE PEGylated ADCs, such as those exemplified herein, aparticularly preferred average drug load is about 8. In exemplaryembodiments, the drug-linkers are conjugated to the cysteine residues ofthe reduced inter-chain disulfides. In some aspects, the actual drugload for individual antibody molecules in the population ofantibody-drug conjugate compounds is from 1 to 10 (or from 6 to 10 orfrom 6 to 8) with a predominant drug loading of 8. A higher drug loadcan be achieved, for example, if, in addition to the interchaindisulfides, drug-linker is conjugated to introduced cysteine residues(such as a cysteine residue introduced at position 239, according to theEU index).

Exemplary ADCs include the following:

or a pharmaceutically acceptable salt thereof wherein n ranges from 8 to36 and most preferably ranges from 8 to 14 (most preferably 12), R^(PR)is hydrogen or a protecting group, e.g., acid labile protecting group,e.g., BOC, R²¹ is a capping unit for the polyethylene glycol moiety,preferably-CH₃ or —CH₂CH₂COH, Ab represents an anti-αVβ6 antibody and prepresents an integer ranging from 1 to 16, preferably 1 to 14, 6 to 12,6 to 10, or 8 to 10 when referring to individual antibody molecules orto an average drug load of from about 4 or about 6 to about 14,preferably about 8 when referring to a population of antibody molecules.

As noted above, the PEG (polyethylene glycol) portion of the drug linkercan range from 8 to 36, however, it has been found that a PEG of 12ethylene oxide units is particularly preferably. It has been found thatlonger PEG chains can result in slower clearance whereas shorter PEGchains can result in diminished activity. Accordingly, the subscript nin all of the embodiments above is preferably 8 to 14, 8 to 12, 10 to 12or 10 to 14 and is most preferably 12.

Polydisperse PEGS, monodisperse PEGS and discrete PEGs can be used tomake the PEGylated antibody drug conjugates of the present invention.Polydisperse PEGs are a heterogenous mixture of sizes and molecularweights whereas monodisperse PEGs are typically purified fromheterogenous mixtures and are therefore provide a single chain lengthand molecular weight. Preferred PEG Units are discrete PEGs, compoundsthat are synthesized in step-wise fashion and not via a polymerizationprocess. Discrete PEGs provide a single molecule with defined andspecified chain length. As with the subscript “p”, when referring topopulations of antibody-drug conjugates, the value for the subscript “n”can be an average number and can be an integer or non-integer number.

In preferred embodiments, covalent attachment of the antibody to thedrug-linker is accomplished through a sulfhydryl functional group of theantibody interacting with a maleimide functional group of a drug linkerto form a thio-substituted succinimide. The sulfhydryl functional groupcan be present on the Ligand Unit in the Ligand's natural state, forexample, in a naturally-occurring residue (inter-chain disulfideresides), or can be introduced into the Ligand via chemical modificationor by biological engineering, or a combination of the two. It will beunderstood that an antibody-substituted succinimide may exist inhydrolyzed form(s). For example, in preferred embodiments, an ADC iscomprised of a succinimide moiety that when bonded to the antibody isrepresented by the structure of:

or is comprised of its corresponding acid-amide moiety that when bondedto the antibody is represented by the structure of:

The wavy line indicates linkage to the remainder of the drug-linker.

In some embodiments, an anti-αvβ6 antibody of the invention isconjugated to monomethyl auristatin E via a MDpr-PEG(12)-gluc linkerforming an antibody-drug conjugate having the structure:

or a pharmaceutically acceptable salt thereof wherein n ranges from 8 to36 and most preferably ranges from 8 to 14 (most preferably 12), R^(PR)is hydrogen or a protecting group, e.g., acid labile protecting group,e.g., BOC. R²¹ is a capping unit for the polyethylene glycol moiety,preferably-CH₃ or —CH₂CH₂CO₂H, Ab represents an anti-αVβ6 antibody and prepresents an integer ranging from 1 to 16, preferably 1 to 14, 6 to 12,6 to 10, or 8 to 10 when referring to individual antibody molecules orto an average drug load of from about 4 or about 6 to about 1.4,preferably about 8 when referring to a population of antibody molecules.

Exemplary antibody-drug conjugates also include camptothecin basedantibody-drug conjugates (i.e., the drug component is a camptothecindrug). Camptothecins are topoisomerase inhibitors that have been shownto have anticancer activity. Typically the camptothecin basedantibody-drug conjugate comprises a linker between the camptothecin drugand the anti-αvβ6 antibody. The linker can be, for example, a cleavablelinker (e.g., a peptidyl linker, a carbohydrate linker) or anon-cleavable linker (e.g., linker released by degradation of theantibody). The synthesis and structure of exemplary camptothecindrug-linkers is described in PCT/US19/025968 (filed Apr. 5, 2019), whichis incorporated herein by reference in its entirety and for allpurposes.

Exemplary anti-αvβ6 antibody drug conjugates include camptothecinantibody drug conjugates as follows wherein p represents the drug loadand Ab represents the anti-αvβ6 antibody:

In some embodiments, the camptothecin ADC has the formula (IC):

or a pharmaceutically acceptable salt thereof;

wherein

Ab is an anti-αvβ6 antibody;

y is 1, 2, 3, or 4, or is 1 or 4; and

z is an integer from 2 to 12, or is 2, 4, 8, or 12;

and p is 1-16.

In some aspect of these embodiments, p is 2, 3, 4, 5, 6, 7, 8, 9, or 10.In some aspect, p is 2, 4 or 8.

In some embodiments, the camptothecin ADC has the formula:

or a pharmaceutically acceptable salt thereof;

wherein p is 2, 4, or 8, preferably p is 8.

In some embodiments, the camptothecin ADC has the formula:

or a pharmaceutically acceptable salt thereof;

wherein p is 2, 4, or 8, preferably p is 8.

In some embodiments, the camptothecin drug-linker has the formula:

or a pharmaceutically acceptable salt thereof;

wherein

y is 1, 2, 3, or 4, or is 1 or 4; and

z is an integer from 2 to 12, or is 2, 4, 8, or 12.

In some embodiments, the camptothecin drug-linker has the formula:

MP-PEG8-VKG-CAMPTOTHECIN

In some embodiments, the camptothecin drug-linker has the formula:

MP-PEG4-VKG-CAMPTOTHECIN

In some embodiments, the camptothecin drug-linker has the formula:

MP-PEG12-VKG-CAMPTOTHECIN

Other exemplary antibody-drug conjugates include maytansinoidantibody-drug conjugates (i.e., the drug component is a maytansinoiddrug), and benzodiazepine antibody drug conjugates (i.e., the drugcomponent is a benzodiazepine (e.g., pyrrolo[1,4]benzodiazepine dimers(PBD dimer), indolinobenzodiazepine dimers, andoxazolidinobenzodiazepine dimers)).

In some embodiments, a PBD dimer for use in the present invention isrepresented by formula I. The preferred stereochemistry of the PBD dimeris as shown in formula Ia:

or a pharmaceutically salt, solvate, or solvate of the salt; wherein thesubscript n is 1, or 3.

Solvates of formula (I) and (Ia) are typically formed from addition ofwater or alcoholic solvent across the imine functional group of one orboth PBD monomers to form carbinolamine(s) and/or carbinolamine ethers.For example, at the N10-C11 position, there can be an imine (N═C), acarbinolamine(NH—CH(OH)), or a carbinolamine ether (NH—CH(OMe)) asrepresented by formulas I′ and Ia′ below:

wherein either:(a) R¹⁰ is H, and R¹¹ is OH or OR^(A), where R^(A) is saturated C₁₋₄alkyl (preferably methyl); or(b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogenand carbon atoms to which they are bound; orc) one of R¹⁰ is H, and R¹¹ is OH or OR^(A), where R^(A) is saturatedC₁₋₄ alkyl (preferably methyl); and the other of R¹⁰ and R¹¹ form anitrogen-carbon double bond between the nitrogen and carbon atoms towhich they are bound.

The PBD dimer of formula I or 1a (or a pharmaceutically salt, solvate,or solvate of the salt thereof) is typically linked to the antibody viaa Linker Unit, LU. The Linker Unit acts to release the PBD dimer offormula I or 1a (or a pharmaceutically salt, solvate, or solvate of thesalt thereof) at the target site (e.g., inside the cancer cell). A PBDdrug-linker compound for use in the present invention is representedbelow by formula II (preferred stereochemistry as shown in Ila) whereinLU is a Linker Unit. The Linker Unit can be, for example, a cleavablepeptide Linker Unit (e.g., a linker comprising the valine-alaninepeptide) or a cleavable disulfide Linker Unit:

or a pharmaceutically salt, solvate, or solvate of the salt; wherein thesubscript n is 1 or 3.

A preferred PBD drug-linker compound for use in the present invention isrepresented by Formula III below:

or a pharmaceutically salt, solvate or solvate of the salt; wherein thesubscript n is 1 or 3 and the subscript m is an integer from 2 to 5.

The PBD drug-linker is conjugated to an anti-αvβ6 antibody to produce aαvβ6 targeted antibody-drug conjugate. For example, the antibody can beconjugated to a drug-linker of formula II or formula III. An exemplaryαvβ6 targeted antibody-drug conjugate is shown below in formulas IV,IVa, and IVb:

or a pharmaceutically salt, solvate, or solvate of the salt; wherein thesubscript n is 1 or 3; the subscript m is an integer from 2 to 5; andthe subscript p is from 1 to 4.

Useful classes of cytotoxic agents to conjugate to anti-αvβ6 antibodiesinclude, for example, antitubulin agents, DNA minor groove bindingagents, DNA replication inhibitors, chemotherapy sensitizers, or thelike. Other exemplary classes of cytotoxic agents includeanthracyclines, auristatins, camptothecins, duocarmycins, etoposides,maytansinoids and vinca alkaloids. Some exemplary cytotoxic agentsinclude auristatins (e.g., auristatin T, auristatin E, AFP, monomethylauristatin F (MMAF), lipophilic monomethyl aurstatin F, monomethylauristatin E (MMAE)), DNA minor groove binders (e.g., enediynes andlexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel),vinca alkaloids, nicotinamide phosphoribosyltranferase inhibitor(NAMPTi), tubulysin M, doxorubicin, morpholino-doxorubicin, andcyanomorpholino-doxorubicin.

The cytotoxic agent can be a chemotherapeutic such as, for example,doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate,mitomycin C or etoposide. The agent can also be a CC-1065 analogue,calicheamicin, maytansine, an analog of dolastatin 10, rhizoxin, orpalytoxin.

The cytotoxic agent can also be an auristatin. The auristatin can be anauristatin E derivative is, e.g., an ester formed between auristatin Eand a keto acid. For example, auristatin E can be reacted withparaacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB,respectively. Other typical auristatins include auristatin T, AFP, MMAF,and MMAE. The synthesis and structure of various auristatins aredescribed in, for example, US 2005-0238649 and US2006-0074008.

The cytotoxic agent can be a DNA minor groove binding agent. (See, e.g.,U.S. Pat. No. 6,130,237.) For example, the minor groove binding agentcan be a CBI compound or an enediyne (e.g., calicheamicin).

The cytotoxic or cytostatic agent can be an anti-tubulin agent. Examplesof anti-tubulin agents include taxanes (e.g., Taxol® (paclitaxel),Taxotere® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g.,vincristine, vinblastine, vindesine, and vinorelbine), and auristatins(e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Exemplary auristatinsare shown below in formulae III-XIII Other suitable antitubulin agentsinclude, for example, baccatin derivatives, taxane analogs (e.g.,epothilone A and B), nocodazole, colchicine and colcimid, estramustine,cryptophysins, cemadotin, maytansinoids, combretastatins, discodermoideand eleuthrobin.

The cytotoxic agent can be a maytansinoid, another group of anti-tubulinagents (e.g., DM1, DM2, DM3, DM4). For example, the maytansinoid can bemaytansine or a maytansine containing drug linker such as DM-1 or DM-4(ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.)

VIII. Therapeutic Applications

The antibodies of the invention, alone or as anti-αvβ6 antibody-drugconjugates thereof, can be used to treat cancer. Some such cancers showdetectable levels of αvβ6 measured at either the protein (e.g., byimmunoassay using one of the exemplified antibodies) or mRNA level. Somesuch cancers show elevated levels of αvβ6 relative to noncanceroustissue of the same type, preferably from the same patient. An exemplarylevel of αvβ6 on cancer cells amenable to treatment is 5000-500,000 αvβ6molecules per cell, although higher or lower levels can be treated.Optionally, a level of αvβ6 in a cancer is measured before performingtreatment.

Examples of cancers associated with αvβ6 expression and amenable totreatment include non-small cell lung cancer (NSCLC) (squamous andadeno), head and neck cancer (including head and neck squamouscarcinoma), esophageal cancer, breast cancer (including breast invasivecarcinoma), ovarian cancer, bladder cancer (including urothelialcarcinoma), skin cancer (squamous cell carcinoma, or SCC), renal cancer(including renal clear cell, renal papillary cell, and kidneychromophobe), cervical cancer, gastric cancer, prostate cancer(including prostate adenocarcinoma), endometrial cancer (includinguterine carcinosarcoma and uterine corpus endometrial), rectumadenocarcinoma, thyroid carcinoma, colon adenocarcinoma, stomachadenocarcinoma, and pancreatic cancer (including pancreaticadenocarcinoma). In some embodiments, the antibodies or antibody-drugconjugates of the invention are used in methods of treating NSCLC. Insome embodiments, the antibodies or antibody-drug conjugates of theinvention are used in methods of treating head and neck cancer. In someembodiments, the antibodies or antibody-drug conjugates of the inventionare used in methods of treating skin cancer. In some embodiments, theantibodies or antibody-drug conjugates of the invention are used inmethods of treating esophageal cancer. In some embodiments, theantibodies or antibody-drug conjugates of the invention are used inmethods of treating breast cancer. In some embodiments, the antibodiesor antibody-drug conjugates of the invention are used in methods oftreating ovarian cancer. In some embodiments, the antibodies orantibody-drug conjugates of the invention are used in methods oftreating bladder cancer. In some embodiments, the antibodies orantibody-drug conjugates of the invention are used in methods oftreating cervical cancer. In some embodiments, the antibodies orantibody-drug conjugates of the invention are used in methods oftreating gastric cancer. In some embodiments, the antibodies orantibody-drug conjugates of the invention are used in methods oftreating renal cancer. In some embodiments, the antibodies orantibody-drug conjugates of the invention are used in methods oftreating endometrial cancer. In some embodiments, the antibodies orantibody-drug conjugates of the invention are used in methods oftreating stomach cancer. In some embodiments, the antibodies orantibody-drug conjugates of the invention are used in methods oftreating pancreatic cancer. The treatment can be applied to patientshaving primary or metastatic tumors of these kinds. The treatment canalso be applied to patients who are refractory to conventionaltreatments, or who have relapsed following a response to suchtreatments.

Antibodies of the present invention, such as humanized antibodies, aloneor as conjugates thereof, are administered in an effective regimemeaning a dosage, route of administration and frequency ofadministration that delays the onset, reduces the severity, inhibitsfurther deterioration, and/or ameliorates at least one sign or symptomof cancer. If a patient is already suffering from cancer, the regime canbe referred to as a therapeutically effective regime. If the patient isat elevated risk of the caner relative to the general population but isnot yet experiencing symptoms, the regime can be referred to as aprophylactically effective regime. In some instances, therapeutic orprophylactic efficacy can be observed in an individual patient relativeto historical controls or past experience in the same patient. In otherinstances, therapeutic or prophylactic efficacy can be demonstrated in apreclinical or clinical trial in a population of treated patientsrelative to a control population of untreated patients.

Exemplary dosages for a monoclonal antibody are 0.1 mg/kg to 50 mg/kg ofthe patient's body weight, more typically 1 mg/kg to 30 mg/kg, 1 mg/kgto 20 mg/kg, 1 mg/kg to 15 mg/kg, 1 mg/kg to 12 mg/kg, or 1 mg/kg to 10mg/kg 1, or 2 mg/kg to 30 mg/kg, 2 mg/kg to 20 mg/kg, 2 mg/kg to 15mg/kg, 2 mg/kg to 12 mg/kg, or 2 mg/kg to 10 mg/kg, or 3 mg/kg to 30mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to 15 mg/kg, 3 mg/kg to 12 mg/kg, or3 mg/kg to 10 mg/kg. Exemplary dosages for a monoclonal antibody orantibody drug conjugates thereof are 1 mg/kg to 7.5 mg/kg, or 2 mg/kg to7.5 mg/kg or 3 mg/kg to 7.5 mg/kg of the subject's body weight, or0.1-20, or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 mg/kg) or 10-1500 or 200-1500 mg as a fixed dosage. In somemethods, the patient is administered a dose of at least 1.5 mg/kg, atleast 2 mg/kg or at least 3 mg/kg, administered once every three weeksor greater. The dosage depends on the frequency of administration,condition of the patient and response to prior treatment, if any,whether the treatment is prophylactic or therapeutic and whether thedisorder is acute or chronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous,intra-arterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Administration can also be localizeddirectly into a tumor. Administration into the systemic circulation byintravenous or subcutaneous administration is preferred. Intravenousadministration can be, for example, by infusion over a period such as30-90 min or by a single bolus injection.

The frequency of administration depends on the half-life of the antibodyor conjugate in the circulation, the condition of the patient and theroute of administration among other factors. The frequency can be daily,weekly, monthly, quarterly, or at irregular intervals in response tochanges in the patient's condition or progression of the cancer beingtreated. An exemplary frequency for intravenous administration isbetween twice a week and quarterly over a continuous course oftreatment, although more or less frequent dosing is also possible. Otherexemplary frequencies for intravenous administration are between weeklyor three out of every four weeks over a continuous course of treatment,although more or less frequent dosing is also possible. For subcutaneousadministration, an exemplary dosing frequency is daily to monthly,although more or less frequent dosing is also possible.

The number of dosages administered depends on the nature of the cancer(e.g., whether presenting acute or chronic symptoms) and the response ofthe disorder to the treatment. For acute disorders or acuteexacerbations of a chronic disorder between 1 and 10 doses are oftensufficient. Sometimes a single bolus dose, optionally in divided form,is sufficient for an acute disorder or acute exacerbation of a chronicdisorder. Treatment can be repeated for recurrence of an acute disorderor acute exacerbation. For chronic disorders, an antibody can beadministered at regular intervals, e.g., weekly, fortnightly, monthly,quarterly, every six months for at least 1, 5 or 10 years, or the lifeof the patient.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. The concentration of antibody in a liquid formulation can bee.g., 1-100 mg/ml, such as 10 mg/ml.

Treatment with antibodies of the invention can be combined withchemotherapy, radiation, stem cell treatment, surgery other treatmentseffective against the disorder being treated. Useful classes of otheragents that can be administered with antibodies and antibody-drugconjugates to αvβ6 as described herein include, for example, antibodiesto other receptors expressed on cancerous cells, antitubulin agents(e.g., auristatins), DNA minor groove binders, DNA replicationinhibitors, alkylating agents (e.g., platinum complexes such ascisplatin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, and the like.

Treatment with the anti-αvβ6 antibody or antibody-drug conjugate,optionally in combination with any of the other agents or regimesdescribed above alone or as an antibody drug conjugate, can increase themedian progression-free survival or overall survival time of patientswith tumors (e.g., non-small cell lung cancer (NSCLC) (squamous andadeno), head and neck cancer (including head and neck squamouscarcinoma), esophageal cancer, breast cancer (including breast invasivecarcinoma), ovarian cancer, bladder cancer (including urothelialcarcinoma), skin cancer (squamous cell carcinoma, or SCC), renal cancer(including renal clear cell, renal papillary cell, and kidneychromophobe), cervical cancer, gastric cancer, prostate cancer(including prostate adenocarcinoma), endometrial cancer (includinguterine carcinosarcoma and uterine corpus endometrial), rectumadenocarcinoma, thyroid carcinoma, colon adenocarcinoma, stomachadenocarcinoma, and pancreatic cancer (including pancreaticadenocarcinoma)), especially when relapsed or refractory, by at least30% or 40% but preferably 50%, 60% to 70% or even 100% or longer,compared to the same treatment (e.g., chemotherapy) but without ananti-αvβ6 antibody alone or as a conjugate. In addition oralternatively, treatment (e.g., standard chemotherapy) including theanti-αvβ6 antibody alone or as a conjugate can increase the completeresponse rate, partial response rate, or objective response rate(complete+partial) of patients with tumors by at least 30% or 40% butpreferably 50%, 60% to 70% or even 100% compared to the same treatment(e.g., chemotherapy) but without the anti-αvβ6 antibody alone or as aconjugate.

Typically, in a clinical trial (e.g., a phase II, phase or phase IIItrial), the aforementioned increases in median progression-free survivaland/or response rate of the patients treated with standard therapy plusthe anti-αvβ6 antibody alone or as conjugate, relative to the controlgroup of patients receiving standard therapy alone (or plus placebo),are statistically significant, for example at the p=0.05 or 0.01 or even0.001 level. The complete and partial response rates are determined byobjective criteria commonly used in clinical trials for cancer, e.g., aslisted or accepted by the National Cancer Institute and/or Food and DrugAdministration.

IX. Articles of Manufacture and Kits

In another aspect, an article of manufacture or kit is provided whichcomprises an anti-αvβ6 antibody or anti-αvβ6 antibody-drug conjugatedescribed herein. The article of manufacture or kit may further compriseinstructions for use of the anti-αvβ6 antibody or anti-αvβ6antibody-drug conjugate described herein in the methods of theinvention. Thus, in certain embodiments, the article of manufacture orkit comprises instructions for the use of an anti-αvβ6 antibody oranti-αvβ6 antibody-drug conjugate described herein in methods fortreating cancer (e.g., non-small cell lung cancer (NSCLC) (squamous andadeno), head and neck cancer (including head and neck squamouscarcinoma), esophageal cancer, breast cancer (including breast invasivecarcinoma), ovarian cancer, bladder cancer (including urothelialcarcinoma), skin cancer (squamous cell carcinoma, or SCC), renal cancer(including renal clear cell, renal papillary cell, and kidneychromophobe), cervical cancer, gastric cancer, prostate cancer(including prostate adenocarcinoma), endometrial cancer (includinguterine carcinosarcoma and uterine corpus endometrial), rectumadenocarcinoma, thyroid carcinoma, colon adenocarcinoma, stomachadenocarcinoma, and pancreatic cancer (including pancreaticadenocarcinoma)) in a subject comprising administering to the subject aneffective amount of an anti-αvβ6 antibody or anti-αvβ6 antibody-drugconjugate described herein. In some embodiments, the cancer is NSCLC. Insome embodiments, the cancer is head and neck cancer. In someembodiments, the cancer is esophageal cancer. In some embodiments, thecancer is breast cancer. In some embodiments, the cancer is ovariancancer. In some embodiments, the antibodies or antibody-drug conjugatesof the invention are used in methods of treating renal cancer. In someembodiments, the antibodies or antibody-drug conjugates of the inventionare used in methods of treating endometrial cancer. In some embodiments,the antibodies or antibody-drug conjugates of the invention are used inmethods of treating stomach cancer. In some embodiments, the cancer isbladder cancer. In some embodiments, the cancer is skin cancer. In someembodiments, the cancer is cervical cancer. In some embodiments, thecancer is gastric cancer. In some embodiments, the cancer is pancreaticcancer. In some embodiments, the subject is a human.

The article of manufacture or kit may further comprise a container.Suitable containers include, for example, bottles, vials (e.g., dualchamber vials), syringes (such as single or dual chamber syringes) andtest tubes. In some embodiments, the container is a vial. The containermay be formed from a variety of materials such as glass or plastic. Thecontainer holds the formulation.

The article of manufacture or kit may further comprise a label or apackage insert, which is on or associated with the container, mayindicate directions for reconstitution and/or use of the formulation.The label or package insert may further indicate that the formulation isuseful or intended for subcutaneous, intravenous (e.g., intravenousinfusion), or other modes of administration for treating cancer in asubject (e.g., non-small cell lung cancer (NSCLC) (squamous and adeno),head and neck cancer (including head and neck squamous carcinoma),esophageal cancer, breast cancer (including breast invasive carcinoma),ovarian cancer, bladder cancer (including urothelial carcinoma), skincancer (squamous cell carcinoma, or SCC), renal cancer (including renalclear cell, renal papillary cell, and kidney chromophobe), cervicalcancer, gastric cancer, prostate cancer (including prostateadenocarcinoma), endometrial cancer (including uterine carcinosarcomaand uterine corpus endometrial), rectum adenocarcinoma, thyroidcarcinoma, colon adenocarcinoma, stomach adenocarcinoma, and pancreaticcancer (including pancreatic adenocarcinoma)). The container holding theformulation may be a single-use vial or a multi-use vial, which allowsfor repeat administrations of the reconstituted formulation. The articleof manufacture or kit may further comprise a second container comprisinga suitable diluent. The article of manufacture or kit may furtherinclude other materials desirable from a commercial, therapeutic, anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

The article of manufacture or kit herein optionally further comprises acontainer comprising a second medicament, wherein the anti-αvβ6 antibodyor anti-αvβ6 antibody-drug conjugate is a first medicament, and whicharticle or kit further comprises instructions on the label or packageinsert for treating the subject with the second medicament, in aneffective amount. In some embodiments, the second medicament is foreliminating or reducing the severity of one or more adverse events.

In some embodiments, the anti-αvβ6 antibody or anti-αvβ6 antibody-drugconjugate is present in the container as a lyophilized powder. In someembodiments, the lyophilized powder is in a hermetically sealedcontainer, such as a vial, an ampoule or sachette, indicating thequantity of the active agent. Where the pharmaceutical is administeredby injection, an ampoule of sterile water for injection or saline canbe, for example, provided, optionally as part of the kit, so that theingredients can be mixed prior to administration. Such kits can furtherinclude, if desired, one or more of various conventional pharmaceuticalcomponents, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Printedinstructions, either as inserts or as labels, indicating quantities ofthe components to be administered, guidelines for administration, and/orguidelines for mixing the components can also be included in the kit.

X. Other Applications

The anti-αvβ6 antibodies described herein, such as humanized anti-αvβ6,antibodies can be used for detecting αvβ6 in the context of clinicaldiagnosis or treatment or in research. Expression of αvβ6 on a cancerprovides an indication that the cancer is amenable to treatment with theantibodies of the present invention. The antibodies can also be sold asresearch reagents for laboratory research in detecting cells bearingαvβ6 and their response to various stimuli. In such uses, monoclonalantibodies can be labeled with fluorescent molecules, spin-labeledmolecules, enzymes or radioisotypes, and can be provided in the form ofkit with all the necessary reagents to perform the assay for αvβ6. Theantibodies described herein, can be used to detect αvβ6 proteinexpression and determine whether a cancer is amenable to treatment withαvβ6 ADCs.

All patent filings, website, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES

Materials

Cell lines described in the following examples were maintained inculture according to the conditions specified by the American TypeCulture Collection (ATCC), the National Cancer Institute (NCI) or theDeutsche Sammlung von Mikroorganismen and Zellkulturen GmbH,Braunschweig, Germany (DMSZ). SW780 cell lines, Detroit 562 cell lines,HPAFII cell lines, and BxPC3 cell lines were obtained from ATCC.FreeStyle™ 293-F (InVitrogen Corp) human epithelial kidney cells andcorresponding transfectants were maintained as described by themanufacterer. Cell culture reagents were obtained from Invitrogen Corp.(Carlsbad, Calif.), Molecular Devices (Sunnydale, Calif.) and othersuppliers. Secondary antibody reagents were purchased from JacksonImmunoResearch Laboratories (West Grove, Pa.). Recombinant αvβ1, αvβ3αvβ5, αvβ6, and αvβ8 were purchased from R&D Systems (Minneapolis,Minn.). FreeStyle™ 293-F cells express endogenous integrin αv and theywere stably transfected with a full length cDNA encoding human,cynomolgus or murine integrin β6 to generate HEK293F:huβ6,HEK293F:cynoβ6 and HEK293F:muβ6 cell lines, respectively. HEK293F cellstransfected with the empty vector (HEK293F:vector) were used as anegative control. Mouse 3T3 and FDC-P1 cells that express endogenousmouse integrin av were transfected with a full-length cDNA clone forhuman and mouse integrin β6 to generate 3T3:huβ6 and FDC-P1:muβ6,respectively.

Methodologies:

Generation of 2A2 Antibody

ICR (CD-1) mice were immunized three times with intraperitonealinjections of ˜5×10⁶ 3 T3:huβ6 transfectants. Three days prior tofusion, mice received a final injection of purified recombinant humanαvβ6 that was given intravenously (6 ug) and intraperitoneally (30 ug).Lymphocytes harvested from spleen and lymph nodes were fused toP3X63Ag8.653 myeloma cells using polyethylene glycol. Fused cells wererecovered overnight in hybridoma growth media (IMDM containing 4 mMglutamine, 10% Fetal Clone I, 10% Cloning Factor andPenicillin/Streptomycin). Following recovery, cells were spun down andthen plated in semi-solid media. Semi-solid media consisted ofCloneMatrix media supplemented with hybridoma growth media plus HAT forhybridoma selection and CloneDetect for IgG-production. Hybridomas wereincubated for 10 days at 37° C. At day 10, IgG producing hybridomaclones were picked using a ClonePixFL (Molecular Devices) andtransferred to 96-well plates containing IgG-depleted hybridoma growthmedia plus HT. Hybridoma culture supernatants were screened on 293F:huβ6transfectants and positive clones were identified using anAlexifluor-647 labeled secondary antibody for detection. The plates wereread in an FMAT 8200 (Applied Biosystems). Hybridomas that bound to293F:huβ6 and 293F:cynoβ6 but not 293F:vector were expanded for directconjugation to drug-linkers. The directly conjugated antibody panel wastested in binding and cytotoxicity assays.

LAP Blockade ELISA

96-well microtiter plates (Nunc) were coated overnight at 4° C. with 0.3ug/mL recombinant human latency-associated peptide (rhuLAP) (madein-house; Lot 09-19-09DS) in 1×PBS. Following removal of coatingsolution, plates were blocked with 3% BSA in Tris-buffered saline (TBS)for one hour room at temperature and then washed 5× with PBS+0.05%Tween-20 (PBST) prior to use. In a separate conical bottom 96-wellplate, 0.25 ug/mL of recombinant human (rhu) αvβ6-biotin (made in-houserhuαvβ6-biotin, Lot #171030A) was pre-incubated for 1 hour at roomtemperature with increasing concentrations of purified antibody in TBSbuffer containing 1 mM CaCl₂, 1 mM MgCl₂ and 1 mg/mL BSA for 1 hour atroom temperature. The antibody/αvβ6-biotin mixture was then transferredto the LAP-coated plate and incubated at room temperature for 1 hour.Plates were washed as above and incubated with 50 uL/well of peroxidaseconjugated strepatvidin (Jackson ImmunoResearch #016-030-084) diluted1:1000 in TBS+1 mg/mL BSA at room temperature for 1 hour. Bound proteinand signal was detected using TMB (Invitrogen #00-2023) incubated for5.5 minutes and then quenched with 1N H₂SO₄ (Fisher #SA212-1). Plateswere immediately read on plate reader (Molecular Devices Vmax KineticMicroplate Reader) at 450 nm wavelength. Data were exported intoMicrosoft Excel and analyzed using GraphPad Prism v5.03.

Competition Binding Assays—Humanized 2A2 Antibody Variants

Competition binding assays were done using the 293F:huβ6 cell line.0.1×10⁶ antigen expressing cells were aliquoted in each well of a96-well v-bottom plate on ice. The cells were incubated for 1 hour with2 nM AlexaFluor-647 labeled m2A2 and increasing concentrations (from0.03-500 nM) of unlabeled humanized 2A2 variant antibodies in FACSbuffer (Tris-buffered saline, 2% fetal bovine serum, 0.5 mM MnCl₂, 0.02%NaN3). Cells were pelleted and washed 3 times with TBS/FBS. The cellswere pelleted and resuspended in 125 uL of TBS/FBS. Fluorescent signalof binding was detected using a Becton Dickinson Biosciences LSR II (SanJose, Calif.). Percent of saturated fluorescent signal was used todetermine percent labeled humanized 2A2 antibody bound and tosubsequently extrapolate the EC50 by fitting the data to a sigmoidaldose-response curve with variable slope using GraphPad Software(LaJolla, Calif.).

Competition Binding Assays—Human and Cyno Δvβ6

Competition binding assays were done using the 293F:huβ6 andHEK293F:cynoβ6 cell lines. 0.1×10⁶ antigen expressing cells werealiquoted in each well of a 96-well v-bottom plate on ice. The cellswere incubated for 1 hour with 2 nM AlexaFluor-647 labeled humanized 2A2HCLG and increasing concentrations (from 4 pM-1 uM) of unlabeledhumanized 2A2 HCLG antibody and h2A2-Mdpr-PEG(12)-gluc-MMAE(8) in buffer(Tris-buffered saline, 2% fetal bovine serum, 0.5 mM MnCl₂, 0.02% NaN3).Cells were pelleted and washed 3 times with TBS/FBS. The cells werepelleted and resuspended in 125 uL of TBS/FBS. Fluorescent signal ofbinding was detected using a Becton Dickinson Biosciences LSR II (SanJose, Calif.). Percent of saturated fluorescent signal was used todetermine percent labeled humanized 2A2 antibody bound to cells and tosubsequently extrapolate the EC50 by fitting the data to a sigmoidaldose-response curve with variable slope using GraphPad Software(LaJolla, Calif.).

αvβ6 Saturation Binding Assays—Human and Cyno αvβ6

Saturation binding studies were done using the following antigenexpressing cell lines: 293F:huβ6 and 293F:cynoβ6. 0.1×10⁶ antigenexpressing cells were aliquoted per well into a 96-well v-bottom plate.m2A2 and h2A2 HCLG were directly labeled with AlexaFluor-647 and addedto cells at concentrations ranging from 6 pM-340 nM in buffer(Tris-buffered saline, 2% fetal bovine serum, 0.5 mM MnCl₂, 0.02% NaN3).Cell were incubated for 1 hour then pelleted and washed 3 times withTBS. The cells were pelleted and resuspended in 120 uL of TBS.Fluorescent signal of binding was detected using a Becton DickinsonBiosciences LSR II (San Jose, Calif.). The EC50 was calculated usingGraphPad Software (LaJolla, Calif.).

ELISA

96-well Maxisorb plates (Nunc) were coated overnight at 4° C. with 1ug/ml of recombinant human αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8 (R&D Systems,MN) diluted in 50 mM carbonate buffer (Sigma, MO). Plates were washedwith PBS+0.05% Tween 20 (PBS-T). Wash buffer was removed and plates wereblocked for 2 hours at room temperature in TBS blocking buffer (TBS,0.05% Tween 20, 1% BSA). Plates were washed and then incubated for 2hours with humanized 2A2 antibody diluted in TBS binding buffer (TBS,0.05% Tween 20, 1% BSA, 1 mM MnCl₂) at concentrations that ranged from 1pM-67 nM (10 ug/ml). Plates were washed, incubated for 1 hour with1:5000 dilution of anti-human Fc-HRP (Jackson ImmunoResearch, PA),washed, and then incubated with TMB substrate for 5 minutes. Thereaction was stopped with 1 M HCl. Absorbance at 450 nm was read using aFusion HT plate reader (Perkin Elmer, Waltham, Mass.).

Quantitative Flow Cytometric Analysis

Quantification of αvβ6 copy number on the cell surfaces was determinedusing murine αvβ6 mAb as primary antibody and the DAKO QiFiKit flowcytometric indirect assay as described by the manufacturer (DAKO A/S,Glostrup, Denmark) and evaluated with a Becton Dickinson FACScan flowcytometer.

In Vitro Cytotoxicity Assay

Tumor cells were incubated with anti-αvβ6 antibody drug conjugates for96 hours at 37° C. Cell viability was determined using theCellTiter-Glo® luminescent assay (Promega Corporation, Madison, Wis.)and results were measured on an EnVision Multilabel plate reader (PerkinElmer, Waltham, Mass.). Results are reported as IC50, defined as theconcentration that results in half maximal growth inhibition over thecourse of the titration curve.

Production of Antibody-Drug Conjugates

Antibody drug conjugates of the anti-αvβ6 antibodies were prepared asdescribed in US20050238649 and WO2015/057699. The drug linkers vcMMAE(also known as 1006) and mcMMAF are both described in US20050238649,which is incorporated herein by reference for all purposes. The druglinker MDpr-Lys(PEGx)-glucuronide-MMAE linker is described inWO2015/057699, which is incorporated herein by reference for allpurposes.

In Vivo Activity Study

For therapy experiments in cell-line derived xenografts, 5×10⁶ cells(ATCC) were injected subcutaneously into 5-8 female nude (nu/nu) mice(Envigo) for the BxPC3, Detroit 562, HPAF-II, and SW780 studies. Micewere randomly divided into study groups and dosed with test article viaintraperitoneal injection once the tumors reached approximately 100 mm³.Animals were euthanized when tumor volumes reached 500-1000 mm³. Tumorvolume was calculated with the formula (volume=½×length×width×width).Mice showing durable regressions were terminated around day 40-65 afterimplant. In all xenograft studies, no weight loss or treatment-relatedtoxicities were observed for mice treated with any of the test articles.All animal procedures were performed under a protocol approved by theInstitutional Animal Care and Use Committee in a facility accredited bythe Association for Assessment and Accreditation of Laboratory AnimalCare.

In addition to the cell-line derived xenografts, the antitumor activityof an h2A2 vcMMAE ADC against patient-derived xenograft (PDX) models ofnon-small cell lung cancer (NSCLC) was studied using models maintainedby Champions Oncology (Hackensack N.J.). These PDX models include NSCLCsamples of both adeno and squamous histology. These models wereestablished by implantation in immunocompromised mice and allowed togrow to a tumor volume of 150-300 mm³, then mice were randomized intotreatment and control groups and dosed with the h2A2 vcMMAE ornon-binding control h00 vcMMAE ADCs. Mice were dosed with 3 mg/kg of ADCweekly for a total of three doses. Tumor volumes were measured twiceweekly for 28 days after the first dose, or until tumors reach a volumeof 1500 mm³.

The antitumor activity of h2A2 vcMMAE ADC was further evaluated in PDXmodels of ovarian carcinoma models maintained by Champions Oncology(Hackensack N.J.). These models were established by implantation inimmunocompromised mice and allowed to grow to a tumor volume of 150-300mm³, then mice were randomized into treatment and control groups anddosed with the ADC. Mice were dosed with 5 mg/kg of ADC weekly for atotal of three doses. Tumor volumes were measured twice weekly for 28days after the first dose, or until tumors reach a volume of 1500 mm³ upto a maximum of 60 days.

Results

The murine clone m2A2 was selected from the hybridoma panel because itshowed cytotoxic activity as an ADC on multiple αvβ6-positive tumor celllines and it had comparable affinity to human and cynomolgus forms ofthe antigen. The specificity of mouse 2A2 was confirmed in FMAT and flowcytometry binding studies where the antibodies were shown to bind to293F:huβ6 transfectants but not to the αvβ5-positive parental line(293F:vector). Integrin αvβ6 has been shown to be a receptor for RGDsites in fibronectin (Weinacker et al., 1994), tenascin (Prieto et al.,1993), vitronectin (Huang et al. 1998) and latency-associated peptide(LAP) (Munger et al. 1999). Binding of integrin αvβ6 to LAP can inducethe spatially restricted activation of transforming growth factor beta1(TGFβ) (Munger et al. 1999). A latency-associated peptide (LAP) blockadeassay was performed (FIG. 1 ), showing that m2A2 (SG-44.2A2) does notblock LAP, in contrast to anti-αvβ6 antibodies m15H3 (SG-42.15H3) (seeWO2013/123152) and positive control 10D5. This suggests that the 2A2antibody can be delivered independent of ligand binding. The negativecontrol was a non-binding IgG control, and SG-44.8B9, SG-33.20B8,SG-44.32A6, and SG-44.34D6 were other clones selected from the hybridomapanel.

Binding of Mouse Antibody

The EC50 for binding of the murine monoclonal antibody 2A2 wasdetermined for human and cyno αvβ6 by saturation binding studies usinggenetically engineered cell lines (293F:huβ6, 293F:cynoβ6) (FIG. 2 ).The genetically engineered cell lines express endogenous αv which pairswith the recombinant β6 chain to produce a heterodimeric receptorcomposed of endogenous αv and recombinant β6.

Design and Selection of Humanized Antibodies

The starting point or donor antibody for humanization in this example isthe murine 2A2 antibody. Genomic sequences provided by IGHV1-46 andIGHJ4 for the heavy chain and by IGKV1D-33 and IGKJ2 for the light chainwere used.

In humanization, ten positions were identified in the heavy chainframework (H2, H28, H48, H67, H69, H71, H73, H74, H78, H93; FIG. 3 ) andtwo positions were identified in the light chain framework (L69 and L71;FIG. 4 ) at which the human acceptor sequence differed from the donorsequence and that may affect antibody binding as a result of contactingantigen directly, affecting conformation of CDRs or affecting packingbetween heavy and light chains. Four humanized heavy chain variants(vHA, vHB, vHC, vHD; FIG. 3 ) and eight humanized light chain variants(vLA, vLB, vLC, vLD, vLE, vLF, vLG, vLH; FIG. 4 ) were madeincorporating back mutations at different permutations of thesepositions. These back mutations are specified in Tables 1-4. Theremainder of the framework positions are occupied by the residues fromthe human acceptor sequence.

TABLE 1 Humanizing Mutations in h2A2 Heavy Chain Variants HV Exon MurineDonor Human Acceptor Framework Acceptor vH Variant Sequence Residues CDRResidues hvHA IGHV1-46/HJ4 H2, H28, H93 none hvHB IGHV1-46/HJ4 H2, H28,H74, H93 none hvHC IGHV1-46/HJ4 H2, H28, H48, H67, none H69, H71, H73,H78, H93 hvHD IGHV1-46/HJ4 H2, H28, H48, H67, none H69, H71, H73, H74,H78, H93

TABLE 2 Specific Murine Framework Mutations in h2A2 Heavy Chain VariantsVariant 2 28 48 67 69 71 73 74 78 93 % Human hvHA F S T 85.7 hvHB F S PT 84.7 hvHC F S I A L V K A T 79.6 hvHD F S I A L V K P A T 78.6

TABLE 3 Humanizing Mutations in h2A2 Kappa Light Chain Variants KV ExonMurine Donor Human Acceptor Framework Acceptor vK Variant SequenceResidues CDR Residues hvLA IGKV1D-33/KJ2 none none hvLB IGKV1D-33/KJ2L69, L71 none hvLC IGKV1D-33/KJ2 L69, L71 L56 hvLD IGKV1D-33/KJ2 L71none hvLE IGKV1D-33/KJ2 L69, L71 L24 hvLF IGKV1D-33/KJ2 L71 L24, L56hvLG IGKV1D-33/KJ2 L69, L71 L55 hvLH IGKV1D-33/KJ2 L69 none

TABLE 4 Specific Murine Framework Mutations in h2A2 Kappa Light ChainVariants Variant 69 71 % Human hvLA 84.2 hvLB R Y 82.1 hvLC R Y 83.2hvLD Y 83.2 hvLE R Y 83.2 hvLF Y 85.3 hvLG R Y 83.2 hvLH R 83.2

Humanized antibodies were then expressed representing combinatorialpermutations of these humanized heavy and light chain variants. Thecompetition binding curves of the resulting humanized antibody variants(alongside the murine 2A2 antibody and a human-murine chimeric antibody)to human αvβ6 are shown in FIGS. 5-7 . Four humanized variants wereselected for further analysis in an in vitro activity assay. In vitroanticancer activity of humanized variants HCLE, HCLG, HCLH, HALG, andhumanized anti-αvβ6 antibody 15H3-HTLC (each conjugated to adrug-linker. Via, n=12, R^(PR)=H, R²¹=CH₃) was measured usingcytotoxicity assays in four cell lines expressing αvβ6 at a variety oflevels based on quantitative flow cytometry analysis (pancreatic cancerHPAFII and BxPC-3 cells, head and neck cancer Detroit 562 cells, andbladder cancer SW780 cells). The results are shown in Table 5.

TABLE 5 In vitro activity assay (×50, nM) BxPC3 Detroit-562 HAPF-IISW780 HCLE 6 17.6 14.8 7.9 HCLG 5.1 13.2 10 5.2 HCLH 15.5 28.4 28.7 11.6HALG 8.2 21.2 17.9 19.6 m2A2 11.1 19.4 15.6 11.7 h15H3-HTLC 8.4 305 8.913.8

The humanized variant comprising heavy chain variant hvHC and lightchain variant hvLG (h2A2 HCLG) was selected for further study.

Binding of h2A2 HCLG to both human and cyno αvβ6 was confirmed bysaturation binding (FIG. 8 ) that included the parental murine 2A2 as areference to demonstrate comparable binding between it and the humanizedvariant. Binding of h2A2 HCLG to both human and cyno αvβ6 was alsoconfirmed by competition binding against fluorescently labeled murine2A2 (FIG. 9 ). This assay also included binding of an ADC prepared withh2A2 HCLG and the SGD-5088 drug-linker by reducing the antibodyinterchain disulfides and conjugation with eight copies of drug-linker.The conjugation process had no impact on binding to αvβ6 from eitherhuman or cyno. Binding specificity of h2A2 HCLG was also confirmed byELISA in which the antibody bound to recombinant human αvβ6 but notαvβ1, αvβ3, αvβ5, or αvβ8 (FIG. 10 ).

In Vitro Anticancer Activity of h2A2 ADC

In vitro anticancer activity of humanized variant HCLG when conjugatedto vcMMAE was measured using cytotoxicity assays in four cell linesexpressing αvβ6 at a variety of levels based on quantitative flowcytometry analysis (pancreatic cancer HPAFII and BxPC-3 cells, head andneck cancer Detroit 562 cells, and bladder cancer SW780 cells). FIG. 11shows that the humanized 2A2 anti-αvβ6 ADC exhibited cell killingbehavior in these assays.

In Vivo Anticancer Activity of h2A2 ADCs

Using the same four cell lines tested in vitro, the anti-tumor activityof humanized 2A2 HCLG antibody conjugated with vcMMAE (average of 4drugs per antibody) in vivo (Figures. 12-15) was demonstrated.Significant tumor growth delay or tumor regression compared to untreatedand non-binding control ADC was observed. h2A2 HCLG-1006(4) refers toantibody drug conjugates of the HCLG humanized form of the parentalmurine antibody 2A2 having an average of 4 vcMMAE drug linker moleculesper antibody. h00-1006 (4) refers to an antibody drug conjugate of anonbinding control antibody having an average of 4 vcMMAE drug linkermolecules per antibody.

In PDX models of NSCLC, humanized 2A2 HCLG antibody conjugated withvcMMAE (average of 4 drugs per antibody) also showed anti-tumor activity(FIG. 16 ). Significant tumor growth delays or tumor regressionscompared to untreated and non-binding control ADC were observed. h2A2HCLG-1006 (4) refers to antibody drug conjugates of the HCLG humanizedform of the parental murine antibody 2A2 having an average of 4 vcMMAEdrug linker molecules per antibody. h00-1006 (4) refers to an antibodydrug conjugate of a nonbinding control antibody having an average of 4vcMMAE drug linker molecules per antibody.

In PDX models of ovarian carcinoma, humanized 2A2 HCLG antibodyconjugated with vcMMAE (average of 4 drugs per antibody) also showedanti-tumor activity (FIG. 17 ). Significant tumor growth delays orslight tumor reductions compared to untreated tumors were observed. h2A2HCLG-1006 (4) refers to antibody drug conjugates of the HCLG humanizedform of the parental murine antibody 2A2 having an average of 4 vcMMAEdrug linker molecules per antibody.

The antitumor activity of h2A2 HCLG was compared with h15H3 whenconjugated with drug-linker VIa (n=12, R^(PR)=H, R²¹=CH₃) with a DAR of8. The study protocol was the same as described above for cellline-derived xenograft models, using the HPAFII and BxPC3 cell lines.Animals were dosed once at 3 mg/kg with either h2A2 HCLG, h15H3, ornon-targeted control (h00) ADCs (FIG. 18 ). In both models the h2A2 HCLGADCs exhibited durable regressions of the implanted tumors, while theh15H3 ADCs exhibited growth delay.

SEQUENCES SEQ ID NO: 1 - m2A2 vHEFQLQQSGPELVKPGASVKISCKASGYSFTDYNVNWVKQSNGKSLEWIGVINPKYGTTRYNQKFKGKATLTVDKPSSTAYMQLNSLTSEDSAVYYCTRGLNAWDYWGQGASVTVSSSEQ ID NO: 2 - mIGHV1-39 (closest murine germline V-gene)EFQLQQSGPELVKPGASVKISCKASGYSFTDYNMNWVKQSNGKSLEWIGVINPNYGTTSYNQKFKGKATLTVDQSSSTAYMQLNSLTSEDSAVYYCAR SEQ ID NO: 3 - hIGHV1-46/HJ4QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFDYWGQGTLVTVSSSEQ ID NO: 4 - h2A2 vHAQFQLVQSGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQGLEWMGVINPKYGTTRYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTRGLNAWDYWGQGTLVTV SSSEQ ID NO: 5 - h2A2 vHBQFQLVQSGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQGLEWMGVINPKYGTTRYNQKFKGRVTMTRDTPTSTVYMELSSLRSEDTAVYYCTRGLNAWDYWGQGTLVTV SSSEQ ID NO: 6 - h2A2 vHCQFQLVQSGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQGLEWIGVINPKYGTTRYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCTRGLNAWDYWGQGTLVTV SSSEQ ID NO: 7 - h2A2 vHDQFQLVQSGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQGLEWIGVINPKYGTTRYNQKFKGRATLTVDKPTSTAYMELSSLRSEDTAVYYCTRGLNAWDYWGQGTLVTV SSSEQ ID NO: 8 - m2A2 vLDIQMTQSPASLSASVGETVTITCGASENIYGALNWYQRKQGKSPQLLIYGATNLADGMSSRFSGSGSGRQYSFKISSLHPDDVATYYCQNVLTTPYTFGGGTKLEIKSEQ ID NO: 9 - mIGKV12-89 (closest murine germline V-gene)DIQMTQSPASLSASVGETVTITCGASENIYGALNWYQRKQGKSPQLLIYGATNLADGMSSRFSGSGSGRQYSLKISSLHPDDVATYYCQNVLSTP SEQ ID NO: 10 - hIGKV1D-33/KJ2DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLP YTFGQGTKLEIKSEQ ID NO: 11 - h2A2 vLADIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKSEQ ID NO: 12 - h2A2 vLBDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKSEQ ID NO: 13 - h2A2 vLCDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLATGVPSRFSGSGSGRDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKSEQ ID NO: 14 - h2A2 vLDDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKSEQ ID NO: 15 - h2A2 vLEDIQMTQSPSSLSASVGDRVTITCQASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKSEQ ID NO: 16 - h2A2 vLFDIQMTQSPSSLSASVGDRVTITCQASENIYGALNWYQQKPGKAPKLLIYGATNLATGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKSEQ ID NO: 17 - h2A2 vLGDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLEDGVPSRFSGSGSGRDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKSEQ ID NO: 18 - h2A2 vLHDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKSEQ ID NO: 19 - h2A2 HA heavy chainQFQLVQSGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQGLEWMGVINPKYGTTRYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCTRGLNAWDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 20 - h2A2 HB heavy chainQFQLVQSGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQGLEWMGVINPKYGTTRYNQKFKGRVTMTRDTPTSTVYMELSSLRSEDTAVYYCTRGLNAWDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 21 - h2A2 HC heavy chainQFQLVQSGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQGLEWIGVINPKYGTTRYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCTRGLNAWDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 22 - h2A2 HD heavy chainQFQLVQSGAEVKKPGASVKVSCKASGYSFTDYNVNWVRQAPGQGLEWIGVINPKYGTTRYNQKFKGRATLTVDKPTSTAYMELSSLRSEDTAVYYCTRGLNAWDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 23 - h2A2 LA light chainDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 24 - h2A2 LB light chainDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 25 - h2A2 LC light chainDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLATGVPSRFSGSGSGRDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 26 - h2A2 LD light chainDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 27 - h2A2 LE light chainDIQMTQSPSSLSASVGDRVTITCQASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 28 - h2A2 LF light chainDIQMTQSPSSLSASVGDRVTITCQASENIYGALNWYQQKPGKAPKLLIYGATNLATGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 29 - h2A2 LG light chainDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLEDGVPSRFSGSGSGRDYTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 30 - h2A2 LH light chainDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDFTFTISSLQPEDIATYYCQNVLTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 31 - HA, HB, HC, HD CDR1, KABAT DYNVNSEQ ID NO: 32 - HA, HB, HC, HD CDR2, KABAT VINPKYGTTRYNQKFKGSEQ ID NO: 33 - HA, HB, HC, HD CDR3, KABAT GLNAWDYSEQ ID NO: 34 - HA, HB, HC, HD CDR1, IMGT GYSFTDYNSEQ ID NO: 35 - HA, HB, HC, HD CDR2, IMGT INPKYGTTSEQ ID NO: 36 - HA, HB, HC, HD CDR3, IMGT TRGLNAWDYSEQ ID NO: 37 - LA, LB, LC, LD, LG, LH CDR1, KABAT GASENIYGALNSEQ ID NO: 38 - LA, LB, LD, LE, LH CDR2, KABAT GATNLADSEQ ID NO: 39 - LA, LB, LC, LD, LE, LF, LG, LH CDR3, KABAT QNVLTTPYTSEQ ID NO: 40 - LE, LF CDR1, KABAT QASENIYGALNSEQ ID NO: 41 - LC, LF CDR2, KABAT GATNLATSEQ ID NO: 42 - LG CDR2, KABAT GATNLEDSEQ ID NO: 43 - LA, LB, LC, LD, LE, LF, LG, LH CDR1, IMGT ENIYGASEQ ID NO: 44 - LA, LB, LC, LD, LE, LF, LG, LH CDR2, IMGT GATSEQ ID NO: 45 - LA, LB, LC, LD, LE, LF, LG, LH CDR3, IMGT QNVLTTPYT

The invention claimed is:
 1. An isolated anti-αvβ6 antibody, orantigen-binding fragment thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises: (i) a CDR-H1 comprising the amino acidsequence of SEQ ID NO:31; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:32; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:33; and wherein the light chain variable regioncomprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ IDNO:37; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:42;and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39wherein the CDRs are determined by Kabat.
 2. An isolated anti-αvβ6antibody, or antigen-binding fragment thereof, comprising a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises: (i) a CDR-H1 comprising the amino acidsequence of SEQ ID NO:34; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:35; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:36; and wherein the light chain variable regioncomprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ IDNO:43; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:44;and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:45wherein the CDRs are determined by IMGT.
 3. The antibody orantigen-binding fragment of claim 1 or claim 2, wherein the antibody ishumanized.
 4. The antibody or antigen-binding fragment of claim 1 orclaim 2, wherein the heavy chain variable region comprises an amino acidsequence having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 6 and the light chain variable region comprisesan amino acid sequence having at least 90% sequence identity to theamino acid sequence of SEQ ID NO:
 17. 5. The antibody or antigen-bindingfragment of claim 1 or claim 2, wherein the heavy chain variable regioncomprises an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 6 and the light chain variableregion comprises an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:
 17. 6. The antibody orantigen-binding fragment of claim 1 or claim 2, wherein the heavy chainvariable region comprises an amino acid sequence having at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 6 and thelight chain variable region comprises an amino acid sequence having atleast 99% sequence identity to the amino acid sequence of SEQ ID NO: 17.7. The antibody or antigen-binding fragment of claim 1 or claim 2,wherein the heavy chain variable region comprises the amino acidsequence of SEQ ID NO: 6 and the light chain variable region comprisesthe amino acid sequence of SEQ ID NO:17.
 8. The antibody orantigen-binding fragment of claim 3, wherein H2 is occupied by F, H28 isoccupied by S, H48 is occupied by I, H67 is occupied by A, H69 isoccupied by L, H71 is occupied by V, H73 is occupied by K, H78 isoccupied by A, H93 is occupied by T, L69 is occupied by R, and L71 isoccupied by Y, and wherein the numbering is via the Kabat numberingsystem.
 9. The antibody or antigen-binding fragment of claim 1 or claim2, wherein the antibody or antigen-binding fragment comprises a heavychain and a light chain, wherein the heavy chain has an amino acidsequence comprising SEQ ID NO:21 and the light chain has an amino acidsequence comprising SEQ ID NO:29.
 10. The antibody or antigen-bindingfragment of claim 1 or claim 2, wherein the antibody or antigen-bindingfragment is an antigen-binding fragment, and wherein the antigen-bindingfragment is selected from the group consisting of Fab, Fab′, F(ab′)₂,Fab′-SH, Fv, diabody, linear antibody, and single-chain antibodyfragment.
 11. The antibody or antigen-binding fragment of claim 1 orclaim 2, wherein the heavy chain variable region of the antibody isfused to a heavy chain constant region and the light chain variableregion is fused to a light chain constant region.
 12. The antibody orantigen-binding fragment of claim 11, wherein the heavy chain constantregion is of the IgG1 isotype.
 13. An antibody-drug conjugate comprisingthe antibody or antigen-binding fragment of claim 1 or claim 2conjugated to a cytotoxic or cytostatic agent.
 14. The antibody-drugconjugate of claim 13, wherein the antibody or antigen-binding fragmentis conjugated to the cytotoxic or cytostatic agent via a linker.
 15. Theantibody-drug conjugate of claim 13, wherein the cytotoxic or cytostaticagent is a monomethyl auristatin.
 16. The antibody-drug conjugate ofclaim 15, wherein the monomethyl auristatin is monomethyl auristatin E(MMAE).
 17. The antibody-drug conjugate of claim 16, wherein theantibody or antigen binding fragment thereof is conjugated to MMAE viaan enzyme-cleavable linker unit.
 18. The antibody-drug conjugate ofclaim 17, wherein the enzyme-cleavable linker unit comprises a Val-Citlinker.
 19. The antibody-drug conjugate of claim 18, wherein theantibody or antigen binding fragment thereof is conjugated to MMAE via alinker unit that has the formula: -A_(a)-W_(w)—Y_(y)—; wherein -A- is astretcher unit, a is 0 or 1; —W— is an amino acid unit, w is an integerranging from 0 to 12; and —Y— is a spacer unit, y is 0, 1, or
 2. 20. Theantibody-drug conjugate of claim 19, wherein the stretcher unit has thestructure of Formula I below; wherein the amino acid unit is Val-Cit;and wherein the spacer unit is a p-aminobenzyl alcohol (PAB) grouphaving the structure of Formula II below;


21. The antibody-drug conjugate of claim 14, wherein the linker isattached to monomethyl auristatin E and has the structure:

wherein Ab is the antibody or antigen-binding fragment and p denotes anumber from 1 to
 16. 22. The antibody-drug conjugate of claim 21,wherein the average value of p in a population of the antibody-drugconjugate is about
 4. 23. A method of treating cancer in a subject, themethod comprising administering to the subject the antibody-drugconjugate of claim
 13. 24. The method of claim 23, wherein the cancer isselected from the group consisting of non-small cell lung cancer(NSCLC), head and neck cancer, esophageal cancer, breast cancer, ovariancancer, bladder cancer, skin cancer (SCC), ovarian cancer, cervicalcancer, gastric cancer, and pancreatic cancer.
 25. The method of claim23, wherein the cancer is non-small cell lung cancer.
 26. The method ofclaim 23, wherein the cancer is head and neck cancer.
 27. The method ofclaim 23, wherein the antibody or antigen-binding fragment orantibody-drug conjugate is in a pharmaceutical composition comprisingthe antibody-drug conjugate and a pharmaceutically acceptable carrier.28. The method of claim 23, wherein the subject is a human.
 29. Apharmaceutical composition comprising the antibody-drug conjugate ofclaim 13 and one or more agents selected from the group consisting of aphysiologically acceptable carrier, a diluent, and an excipient.
 30. Themethod of claim 23, wherein the antibody-drug conjugate is administeredin combination with radiation or a chemotherapeutic agent.
 31. Anantibody-drug conjugate comprising an anti-αvβ6 antibody conjugated tovcMMAE, wherein the antibody has a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 6 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 17, andwherein the antibody-drug conjugate has the structure:

wherein Ab is the antibody and p denotes a number from 1 to
 16. 32. Anisolated anti-αvβ6 antibody, or antigen-binding fragment thereof,comprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises the amino acidsequence of SEQ ID NO: 6 and the light chain variable region comprisesthe amino acid sequence of SEQ ID NO:17, and wherein the heavy chainvariable region of the antibody is fused to a heavy chain constantregion and the light chain variable region is fused to a light chainconstant region.
 33. The antibody or antigen-binding fragment of claim32, wherein the heavy chain constant region is of the IgG1 isotype. 34.An antibody-drug conjugate comprising an anti-αvβ6 antibody, orantigen-binding fragment thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 6 andthe light chain variable region comprises the amino acid sequence of SEQID NO:17, wherein the antibody or antigen-binding fragment is conjugatedto a cytotoxic or cytostatic agent.
 35. The antibody-drug conjugate ofclaim 21, wherein the average value of p in a population of theantibody-drug conjugates is about 2 to about
 5. 36. The antibody-drugconjugate of claim 31, wherein the average value of p in a population ofthe antibody-drug conjugates is about 2 to about
 5. 37. Theantibody-drug conjugate of claim 31, wherein the average value of p in apopulation of the antibody-drug conjugates is about
 4. 38. Theantibody-drug conjugate of claim 34, wherein the antibody orantigen-binding fragment is conjugated to the cytotoxic or cytostaticagent via a linker.
 39. The antibody-drug conjugate of claim 34, whereinthe cytotoxic or cytostatic agent is a monomethyl auristatin.
 40. Theantibody-drug conjugate of claim 39, wherein the monomethyl auristatinis monomethyl auristatin E (MMAE).
 41. The antibody-drug conjugate ofclaim 40, wherein the antibody or antigen binding fragment thereof isconjugated to MMAE via an enzyme-cleavable linker unit.
 42. Theantibody-drug conjugate of claim 41, wherein the enzyme-cleavable linkerunit comprises a Val-Cit linker.
 43. The antibody-drug conjugate ofclaim 42, wherein the antibody or antigen binding fragment thereof isconjugated to MMAE via a linker unit that has the formula:-A_(a)-W_(w)—Y_(y)—; wherein -A- is a stretcher unit, a is 0 or 1; —W—is an amino acid unit, w is an integer ranging from 0 to 12; and —Y— isa spacer unit, y is 0, 1, or
 2. 44. The antibody-drug conjugate of claim43, wherein the stretcher unit has the structure of Formula I below;wherein the amino acid unit is Val-Cit; and wherein the spacer unit is ap-aminobenzyl alcohol (PAB) group having the structure of Formula IIbelow;


45. An antibody-drug conjugate comprising an anti-αvβ6 antibodycomprising a heavy chain and a light chain, wherein the heavy chain hasan amino acid sequence comprising SEQ ID NO:21 and the light chain hasan amino acid sequence comprising SEQ ID NO:29, wherein the antibody isconjugated to a cytotoxic or cytostatic agent.
 46. The antibody-drugconjugate of claim 45, wherein the antibody is conjugated to thecytotoxic or cytostatic agent via a linker.
 47. The antibody-drugconjugate of claim 45, wherein the cytotoxic or cytostatic agent is amonomethyl auristatin.
 48. The antibody-drug conjugate of claim 47,wherein the monomethyl auristatin is monomethyl auristatin E (MMAE). 49.The antibody-drug conjugate of claim 48, wherein the antibody isconjugated to MMAE via an enzyme-cleavable linker unit.
 50. Theantibody-drug conjugate of claim 49, wherein the enzyme-cleavable linkerunit comprises a Val-Cit linker.
 51. The antibody-drug conjugate ofclaim 50, wherein the antibody is conjugated to MMAE via a linker unitthat has the formula: -A_(a)-W_(w)—Y_(y)—; wherein -A- is a stretcherunit, a is 0 or 1; —W— is an amino acid unit, w is an integer rangingfrom 0 to 12; and —Y— is a spacer unit, y is 0, 1, or
 2. 52. Theantibody-drug conjugate of claim 51, wherein the stretcher unit has thestructure of Formula I below; wherein the amino acid unit is Val-Cit;and wherein the spacer unit is a p-aminobenzyl alcohol (PAB) grouphaving the structure of Formula II below;


53. The antibody-drug conjugate of claim 46, wherein the linker isattached to monomethyl auristatin E and has the structure:

wherein Ab is the antibody and p denotes a number from 1 to
 16. 54. Theantibody-drug conjugate of claim 53, wherein the average value of p in apopulation of the antibody-drug conjugates is about 2 to about
 5. 55.The antibody-drug conjugate of claim 53, wherein the average value of pin a population of the antibody-drug conjugate is about
 4. 56. Themethod of claim 23, wherein the cancer is esophageal cancer.
 57. Amethod of treating cancer in a subject, the method comprisingadministering to the subject the antibody-drug conjugate of claim 31.58. The method of claim 57, wherein the cancer is selected from thegroup consisting of non-small cell lung cancer (NSCLC), head and neckcancer, esophageal cancer, breast cancer, ovarian cancer, bladdercancer, skin cancer (SCC), ovarian cancer, cervical cancer, gastriccancer, and pancreatic cancer.
 59. The method of claim 57, wherein thecancer is non-small cell lung cancer.
 60. The method of claim 57,wherein the cancer is head and neck cancer.
 61. The method of claim 57,wherein the cancer is esophageal cancer.
 62. The method of claim 57,wherein the antibody or antigen-binding fragment or antibody-drugconjugate is in a pharmaceutical composition comprising theantibody-drug conjugate and a pharmaceutically acceptable carrier. 63.The method of claim 57, wherein the subject is a human.
 64. The methodof claim 57, wherein the antibody-drug conjugate is administered incombination with radiation or a chemotherapeutic agent.
 65. Apharmaceutical composition comprising the antibody-drug conjugate ofclaim 31 and one or more agents selected from the group consisting of aphysiologically acceptable carrier, a diluent, and an excipient.
 66. Amethod of treating cancer in a subject, the method comprisingadministering to the subject the antibody-drug conjugate of claim 34.67. The method of claim 66, wherein the cancer is selected from thegroup consisting of non-small cell lung cancer (NSCLC), head and neckcancer, esophageal cancer, breast cancer, ovarian cancer, bladdercancer, skin cancer (SCC), ovarian cancer, cervical cancer, gastriccancer, and pancreatic cancer.
 68. The method of claim 66, wherein thecancer is non-small cell lung cancer.
 69. The method of claim 66,wherein the cancer is head and neck cancer.
 70. The method of claim 66,wherein the cancer is esophageal cancer.
 71. The method of claim 66,wherein the antibody or antigen-binding fragment or antibody-drugconjugate is in a pharmaceutical composition comprising theantibody-drug conjugate and a pharmaceutically acceptable carrier. 72.The method of claim 66, wherein the subject is a human.
 73. The methodof claim 66, wherein the antibody-drug conjugate is administered incombination with radiation or a chemotherapeutic agent.
 74. Apharmaceutical composition comprising the antibody-drug conjugate ofclaim 34 and one or more agents selected from the group consisting of aphysiologically acceptable carrier, a diluent, and an excipient.
 75. Amethod of treating cancer in a subject, the method comprisingadministering to the subject the antibody-drug conjugate of claim 45.76. The method of claim 75, wherein the cancer is selected from thegroup consisting of non-small cell lung cancer (NSCLC), head and neckcancer, esophageal cancer, breast cancer, ovarian cancer, bladdercancer, skin cancer (SCC), ovarian cancer, cervical cancer, gastriccancer, and pancreatic cancer.
 77. The method of claim 75, wherein thecancer is non-small cell lung cancer.
 78. The method of claim 75,wherein the cancer is head and neck cancer.
 79. The method of claim 75,wherein the cancer is esophageal cancer.
 80. The method of claim 75,wherein the antibody or antigen-binding fragment or antibody-drugconjugate is in a pharmaceutical composition comprising theantibody-drug conjugate and a pharmaceutically acceptable carrier. 81.The method of claim 75, wherein the subject is a human.
 82. The methodof claim 75, wherein the antibody-drug conjugate is administered incombination with radiation or a chemotherapeutic agent.
 83. Apharmaceutical composition comprising the antibody-drug conjugate ofclaim 45 and one or more agents selected from the group consisting of aphysiologically acceptable carrier, a diluent, and an excipient.
 84. Amethod of treating cancer in a subject, the method comprisingadministering to the subject the antibody-drug conjugate of claim 53.85. The method of claim 84, wherein the cancer is selected from thegroup consisting of non-small cell lung cancer (NSCLC), head and neckcancer, esophageal cancer, breast cancer, ovarian cancer, bladdercancer, skin cancer (SCC), ovarian cancer, cervical cancer, gastriccancer, and pancreatic cancer.
 86. The method of claim 84, wherein thecancer is non-small cell lung cancer.
 87. The method of claim 84,wherein the cancer is head and neck cancer.
 88. The method of claim 84,wherein the cancer is esophageal cancer.
 89. The method of claim 84,wherein the antibody or antigen-binding fragment or antibody-drugconjugate is in a pharmaceutical composition comprising theantibody-drug conjugate and a pharmaceutically acceptable carrier. 90.The method of claim 84, wherein the subject is a human.
 91. The methodof claim 84, wherein the antibody-drug conjugate is administered incombination with radiation or a chemotherapeutic agent.
 92. Apharmaceutical composition comprising the antibody-drug conjugate ofclaim 53 and one or more agents selected from the group consisting of aphysiologically acceptable carrier, a diluent, and an excipient.
 93. Anantibody-drug conjugate comprising an anti-αvβ6 antibody, orantigen-binding fragment thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises: (i) a CDR-H1 comprising the amino acidsequence of SEQ ID NO:31; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:32; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:33; and wherein the light chain variable regioncomprises: (i) a CDR-L1 comprising the amino acid sequence of SEQ IDNO:37; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:42;and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39wherein the CDRs are determined by Kabat, conjugated to a cytotoxic orcytostatic agent via a linker, wherein the linker is attached tomonomethyl auristatin E, and the antibody-drug conjugate has thestructure:

wherein Ab is the antibody or antigen-binding fragment and the averagevalue of p in a population of the antibody-drug conjugate is about 2 toabout
 5. 94. An antibody-drug conjugate comprising an anti-αvβ6antibody, or antigen-binding fragment thereof, comprising a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the amino acid sequence of SEQ ID NO: 6and the light chain variable region comprises the amino acid sequence ofSEQ ID NO:17, conjugated to a cytotoxic or cytostatic agent via alinker, wherein the linker is attached to monomethyl auristatin E andthe antibody-drug conjugate has the structure:

wherein Ab is the antibody or antigen-binding fragment and the averagevalue of p in a population of the antibody-drug conjugate is about 2 toabout
 5. 95. An antibody-drug conjugate comprising an anti-αvβ6antibody, or antigen-binding fragment thereof, comprising a heavy chainand a light chain, wherein the heavy chain has an amino acid sequencecomprising SEQ ID NO:21 and the light chain has an amino acid sequencecomprising SEQ ID NO:29, conjugated to a cytotoxic or cytostatic agentvia a linker, wherein the linker is attached to monomethyl auristatin Eand the antibody-drug conjugate has the structure:

wherein Ab is the antibody or antigen-binding fragment and the averagevalue of p in a population of the antibody-drug conjugate is about 2 toabout
 5. 96. A method of treating cancer in a human subject, the methodcomprising administering to the subject the antibody-drug conjugatecomprising an anti-αvβ6 antibody, or antigen-binding fragment thereof,comprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises: (i) a CDR-H1comprising the amino acid sequence of SEQ ID NO:31; (ii) a CDR-H2comprising the amino acid sequence of SEQ ID NO:32; and (iii) a CDR-H3comprising the amino acid sequence of SEQ ID NO:33; and wherein thelight chain variable region comprises: (i) a CDR-L1 comprising the aminoacid sequence of SEQ ID NO:37; (ii) a CDR-L2 comprising the amino acidsequence of SEQ ID NO:42; and (iii) a CDR-L3 comprising the amino acidsequence of SEQ ID NO:39 wherein the CDRs are determined by Kabat,conjugated to a cytotoxic or cytostatic agent via a linker, wherein thelinker is attached to monomethyl auristatin E, and the antibody-drugconjugate has the structure:

wherein Ab is the antibody or antigen-binding fragment and the averagevalue of p in a population of the antibody-drug conjugate is about 2 toabout
 5. 97. The method of claim 96, wherein the average value of p in apopulation of the antibody-drug conjugate is about
 4. 98. The method ofclaim 96, wherein the cancer is selected from the group consisting ofnon-small cell lung cancer (NSCLC), head and neck cancer, esophagealcancer, breast cancer, ovarian cancer, bladder cancer, skin cancer(SCC), ovarian cancer, cervical cancer, gastric cancer, and pancreaticcancer.
 99. A method of treating cancer in a human subject, the methodcomprising administering to the subject the antibody-drug conjugatecomprising an anti-αvβ6 antibody, or antigen-binding fragment thereof,comprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises the amino acidsequence of SEQ ID NO: 6 and the light chain variable region comprisesthe amino acid sequence of SEQ ID NO:17, conjugated to a cytotoxic orcytostatic agent via a linker, wherein the linker is attached tomonomethyl auristatin E and the antibody-drug conjugate has thestructure:

wherein Ab is the antibody or antigen-binding fragment and the averagevalue of p in a population of the antibody-drug conjugate is about 2 toabout
 5. 100. The method of claim 99, wherein the average value of p ina population of the antibody-drug conjugate is about
 4. 101. The methodof claim 99, wherein the cancer is selected from the group consisting ofnon-small cell lung cancer (NSCLC), head and neck cancer, esophagealcancer, breast cancer, ovarian cancer, bladder cancer, skin cancer(SCC), ovarian cancer, cervical cancer, gastric cancer, and pancreaticcancer.
 102. A method of treating cancer in a human subject, the methodcomprising administering to the subject the antibody-drug conjugatecomprising an anti-αvβ6 antibody, or antigen-binding fragment thereof,comprising a heavy chain and a light chain, wherein the heavy chain hasan amino acid sequence comprising SEQ ID NO:21 and the light chain hasan amino acid sequence comprising SEQ ID NO:29, conjugated to acytotoxic or cytostatic agent via a linker, wherein the linker isattached to monomethyl auristatin E and the antibody-drug conjugate hasthe structure:

wherein Ab is the antibody or antigen-binding fragment and the averagevalue of p in a population of the antibody-drug conjugate is about 2 toabout
 5. 103. The method of claim 102, wherein the average value of p ina population of the antibody-drug conjugate is about
 4. 104. The methodof claim 102, wherein the cancer is selected from the group consistingof non-small cell lung cancer (NSCLC), head and neck cancer, esophagealcancer, breast cancer, ovarian cancer, bladder cancer, skin cancer(SCC), ovarian cancer, cervical cancer, gastric cancer, and pancreaticcancer.